WO2025259938A2

WO2025259938A2 - Methods, compositions, and systems for sample analysis

Info

Publication number: WO2025259938A2
Application number: PCT/US2025/033462
Authority: WO
Inventors: Nathaniel Ryan HENDRICK; Aaron Beaty BEELER; Andrew Martin; Anderson Chen
Original assignee: Prisma Therapeutics Inc; Boston University
Current assignee: Prisma Therapeutics Inc; Boston University
Priority date: 2024-06-13
Filing date: 2025-06-13
Publication date: 2025-12-18
Anticipated expiration: 2026-12-13
Also published as: WO2025259938A3

Abstract

Provided here are methods, systems, compositions, and/or kit polypeptide or sample analysis. Described herein is a method, comprising contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide, comprising a photo-cleavable moiety; and subjecting at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide and a fragment comprising a residue of the terminus of the polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

Description

METHODS, COMPOSITIONS, AND SYSTEMS FOR SAMPLE ANALYSIS

RELATED APPLICATIONS

[0001] This application claims the benefits of and priority to U.S. Provisional Patent Application No. 63/659,695, filed June 13, 2024, the contents of each of which are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Protein sequencing, the determination of the precise order of amino acids within a polypeptide, is a cornerstone of molecular biology and is paramount to understanding a protein’s identity and function. By elucidating the primary structure, the linear order of amino acids, one can predict the protein’s three-dimensional shape and local features in the secondary structure, such as transmembrane and misfolded regions. Thus, identifying a protein's primary structure is incredibly useful for discerning its function and potential role in disease states.

SUMMARY

[0003] In an aspect, the present disclosure provides a method, comprising contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide, comprising a photo-cleavable moiety; and subjecting at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide and a fragment comprising a residue of the terminus of the polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

[0004] In another aspect, the present disclosure provides a method, comprising contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide; and subjecting at least a portion of the first modified polypeptide to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

[0005] In another aspect, the present disclosure provides a method for sample analysis, comprising (a) providing a sample comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is coupled to a first one or more probes and the second polypeptide is coupled to a second one or more probes, (b) detecting (1) one or more signals or signal change from the first one or more probes and (2) one or more signals or signal change from the second one or more probes, (c) contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent, thereby removing at least one amino acid from the first polypeptide and/or from the second polypeptide, wherein the first degradation agent comprises a first photo-cleavable moiety and/or the second degradation agent comprises a second photo-cleavable moiety; and (d) identifying one or more characteristics of the sample.

[0006] In another aspect, the present disclosure provides a compound of Formula I, or a salt, solvate, or a derivative thereof wherein: LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety. [0007] In another aspect, the present disclosure provides a method, comprising (a) providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes, (b) detecting one or more signals or signal change from the one or more probes, and (c) subjecting at least a portion of the polypeptide to (1) a first light comprising a first one or more wavelengths sufficient to remove the one or more probes, and (2) second light comprising a second one or more wavelengths sufficient to remove a terminal amino acid of the polypeptide.

[0008] In another aspect, the present disclosure provides a method, comprising (a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide, and (b) subjecting at least a portion of the first modified polypeptide in a solution to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the solution comprises one or more acids, wherein the second modified polypeptide comprises one or more fewer amino acids than the polypeptide.

[0009] In another aspect, the present disclosure provides a method for analyzing a sample, comprising (a) providing a first polypeptide to a first location on a first support and a second polypeptide to a second location on a second support, wherein the first polypeptide is coupled to first one or more probes, (b) detecting one or more signals or signal change from the one or more probes of the first polypeptide, and (c) selectively subjecting at least a portion of the first polypeptide to light comprising one or more wavelengths sufficient to selectively remove a terminal amino acid of the first polypeptide. [0010] In another aspect, the present disclosure provides a method, comprising (a) detecting one or more signals or signal change from one or more probes coupled to a polypeptide; (b) subjecting at least a portion of the polypeptide to a light condition sufficient to remove a terminal amino acid of the polypeptide; and (c) using the one or more signals or signal change to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.

[0011] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0012] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0013] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which: [0016] FIG. 1 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0017] FIG. 2 shows the mechanism of aqueous N-degradation via guanidinylation (Hamada degradation).

[0018] FIG. 3 shows the method for N-terminal degradation which uses a photocaged amidine (3.29) to perform N-degradation following irradiation.

[0019] FIG. 4 shows conjugation and N-degradation scheme of angiotensin 1 (3.46) using photocaged probe (3.45) in aqueous buffers.

[0020] FIG. 5 shows kinetic timepoint assay of probe (3.45) binding to amine bearing silica beads followed by fhiorophore, AlexaFluor488-NHS.

[0021] FIG. 6 shows pH dependency on reaction rate of N-degradation when R = CH3 (alanine) for (3.17) in Hamada degradation.

[0022] FIG. 7 shows proposed increased cyclization rate of the N-terminal guanidine due to Thorpe-Ingold effect.

[0023] FIG. 8 shows scheme and scope of formation of amidine sulfonates (3.50) from corresponding thioureas (3.49) followed by Nvoc-photocaged amidine sulfonates (3.51).

[0024] FIG. 9A shows ¹ H NMR (500 MHz, d-DMSO) spectrum of (3.60).

[0025] FIG. 9B shows ¹³C NMR (126 MHz, d-DMSO) spectrum of (3.60).

[0026] FIG. 9C shows 'HNMR (500 MHz, d-DMSO) spectrum of (3.61).

[0027] FIG. 9D shows ¹³C NMR (126 MHz, d-DMSO) spectrum of (3.61).

[0028] FIG. 9E shows ¹ H NMR (500 MHz, d-DMSO) spectrum of (3.63).

[0029] FIG. 9F shows ¹³C NMR (126 MHz, d-DMSO) spectrum of (3.63).

[0030] FIG. 9G shows ¹ H NMR (500 MHz, d-DMSO) spectrum of (3.45).

[0031] FIG. 9H shows ¹³C NMR (126 MHz, d-DMSO) spectrum of (3.45).

[0032] FIG. 10 shows a scheme of photocaged amidine probes conjugating to angiotensin 1 and N-degradation of peptide conjugate (above). Results of N-terminal conjugation and N- degradation in aqueous buffers. All reactions were performed at 1 mM angiotensin 1 (3.46) and 10 mM probe (3.51) and results were determined by UPLC-MS.

[0033] FIG. 11 shows kinetic data of photocaged amidine probes reacting with phenylalanine. All reactions were performed at 1 mM probe and 2 mM phenylalanine in pH 9.2 bicarbonate buffer. All measurements were performed by UPLC-MS.

[0034] FIG. 12A shows UPLC-MS spectra of phenylalanine reacting with a probe (3.45) after 120 minutes. [0035] FIG. 12B shows UPLC-MS spectra of phenylalanine reacting with probe (3.60) after 120 minutes.

[0036] FIG. 12C shows UPLC-MS spectra of phenylalanine reacting with probe (3.61) after 120 minutes.

[0037] FIG. 12D shows UPLC-MS spectra of phenylalanine reacting with probe (3.62) after 120 minutes.

[0038] FIG. 13 shows a scheme of peptide conjugation and degradation (above) and peptide fragment scope used for degrader studies (below).

[0039] FIG. 14 shows reaction conversion of N-terminal conjugation of (3.45) at pH 9.2 and photoinduced degradation in pH 13 with different peptide fragments. All reactions were performed with 1 mM peptide and 5 mM (3.45). Analysis was performed by UPLC-MS.

[0040] FIG. 15 shows calculated secondary structures of the EGFR (left) and KRAS (right) fragments calculated by AlphaFold 3.

[0041] FIG. 16 shows scheme of two N-terminal conjugations and degradations of angiotensin 1 with probe (3.45).

[0042] FIG. 17 shows the mechanism of the degradation of polypeptide by degradation agents comprising a photo-cleavable moiety.

[0043] FIG. 18 shows the mechanism of the degradation of polypeptide by degradation agents 3.45.

[0044] FIG. 19 shows scheme and scope of asymmetric bis-biarylated-8-methylthio- BODIPY dyes via an auto-photocatalyzed Meerwein arylation reaction.

DETAILED DESCRIPTION

[0045] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0046] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0047] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0048] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0049] As used herein, the “leaving group” can refercan be an atom or group that is cleaved under the conditions of a substitution reaction. Leaving groups can be, but not limited to, alkane or arylene sulfonyloxy such as methanesulfonyloxy, ethanesulfonyloxy, benzenesulfonyloxy, tosyloxy, thiomethyl, halogen, and/or thienyloxy; dihalogenophosphinoyloxy optionally substituted with isopropyloxy, acyloxy, benzyloxy, and/or the like. In some cases, the leaving group may be HC (O) — COOH or RC (O) — COOH, where R is Ci -Ce Ce alkyl or substituted Ci -C₆ alkyl.

[0050] As used herein the “protecting group”, can refer to a labile chemical moiety which protects reactive groups including without limitation, amino hydroxyl, and/or thiol groups, against undesired reactions. Protecting groups can be used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007. Protecting groups include, e.g. silyl groups such as tertbutyldimethylsilyl (TBDMS), tert-Butyldiphenylsilyl (TBDPS), triisopropyl silyl (TIPS), triisopropyl silyloxymethyl (TOM), triethylsilyl (TES), trimethyl silyl (TMS), or any combination thereof. Protecting groups also include, e.g., a benzyl group, a tosyl group, a triphenylmethane group, a methylthiomethyl ether group, a carbobenzyl oxy group, a p-methoxybenzyl ether (PMB) group, a 9-fluorenylmethyloxycarbonyl (FMOC) group, a pivaloyl group, a tetrahydropyranyl (THP) group, an acetyl group, a benzoyl group, a silyl group, a methyl ether, an ethoxy ethyl, a sulfonamide group, or any combination thereof.

[0051] As used herein, the “electron withdrawing group” can refer to a group that withdraws electron density, such as, for example, from the pi-system of the indeno-fused naphthopyran core structure, or through the sigma-system of a haloalkyl compound. In some cases, an “electron withdrawing group”, as used herein, can be defined as a group having a positive Hammett G_P value, when the group is attached to a carbon participating in an aromatic pi- system, such as the aromatic pi-system of the indeno-fused naphthopyran core. The “Hammett G_P value” can refer to a measurement of the electronic influence, as either an electron-donating or electron- withdrawing influence, of a substituent attached to a carbon participating in an aromatic pi system that is transmitted through the polarizable pi electron system, such as, for example, an aromatic pi electron system. The Hammett G_P value can be a relative measurement comparing the electronic influence of the substituent in the para position of a phenyl ring to the electronic influence of a hydrogen substituted at the para position. In many cases, for aromatic substituents, a negative Hammett G_P value can indicate that a group or substituent donates electron density to another portion of a molecule, while (e.g., acts as an electron-donating group) a positive Hammett G_P value indicates that a group or substituent withdraws electron density from another portion of a molecule (e.g., acts as an electron- withdrawing group). In some cases, Electron-withdrawing groups suitable for use in connection with embodiments of the disclosure may have a Hammett G_P value ranging from about 0.05 to about 0.75. Suitable electronwithdrawing groups may comprise, for example: halogen, such as fluoro (o_p = 0.06), chloro (o_p = 0.23), and bromo (o_p = 0.23); perfluoroalkyl (for example, -CF3, G_P = 0.54) or perfluoroalkoxy (for example, -OCF3, G_P = 0.35). Further suitable electron- withdrawing substituents having Hammett G_P values in the range from about 0.05 to about 0.75 are set forth in “Section 9 Physicochemical Relationships” in Lange 's Handbook of Chemistry, 15^th ed. J. A. Dean, editor, McGraw Hill, 1999, pp 9.1-9.8, the disclosure of which is incorporated herein by reference. In some cases, when referring to the Hammett G value, the subscript “p”, refers to the Hammett G_P value as measured when the group is located at the para position of a phenyl ring of a model system, such as a para-substituted benzoic acid model system.

[0052] As used herein, the “electron donating group” can refer to a group that increases electron density in another portion of a molecule, such as, for example, an alkylamino substituent which donates electron density into an aromatic system. Examples of an “electrondonating group” can include an atom bonded directly to a pi-system of the photochromic material, wherein the atom has at least one lone pair of electrons which are capable of resonance into the pi system of the aromatic ring structure, and/or the group may donate electron density into the pi system by a hyperconjugative effect, such as, for example, an alkyl substituent. In some cases, an “electron donating group”, as used herein, can be defined as a group having a negative Hammett G_P value, when the group is attached to a carbon participating in an aromatic pi system. Example electron donating groups for use with methods and compositions according to the present disclosure include e.g. vinyl, aryl, heteroaryl, amine, alkoxy, and alkyl groups. [0053] “Alkyl” can refer to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from one to fifteen carbon atoms (z.e., C1-C15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (z.e., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (z.e., Ci-Cs alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (z.e., C1-C5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (z.e., Ci- C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (z.e., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (z.e., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (z.e., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (z.e., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (z.e., Cs-Cs alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (z.e., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (z.e., C3-C5 alkyl). In certain embodiments, the alkyl group can comprise 1 -methylethyl (z.w-propyl), 1 -butyl (zz-butyl), 1 -methylpropyl ( ec-butyl), 2- methylpropyl (z.w-butyl), 1,1 -dimethylethyl (tert-butyl), methyl, ethyl, 1 -propyl (zz-propyl), and/or 1 -pentyl (zz-pentyl). The alkyl is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0054] “Alkenyl” can refer to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms (z.e., C2-C12 alkenyl). In certain embodiments, an alkenyl comprises two to eight carbon atoms (z.e., C2-C8 alkenyl). In certain embodiments, an alkenyl comprises two to six carbon atoms (z.e., C2-C6 alkenyl). In other embodiments, an alkenyl comprises two to four carbon atoms (z.e., C2-C4 alkenyl). The alkenyl is attached to the rest of the molecule by a single bond, for example, epent-l-enyl, penta- 1,4-dienyl, thenyl (z.e., vinyl), prop-l-enyl (z.e., allyl), but-l-enyl, and/or the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0055] “Alkynyl” can refer to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and having from two to twelve carbon atoms (z.e., C2-C12 alkynyl). In certain embodiments, an alkynyl comprises two to eight carbon atoms (z.e., C2-C8 alkynyl). In other embodiments, an alkynyl comprises two to six carbon atoms (z.e., C2-C6 alkynyl). In other embodiments, an alkynyl comprises two to four carbon atoms (z.e., C2-C4 alkynyl). The alkynyl is attached to the rest of the molecule by a single bond, for example, pentynyl, hexynyl, ethynyl, propynyl, butynyl, and/or the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0056] Included in the present disclosure are salts, including pharmaceutically acceptable salts, of the compounds described herein. The compounds of the present disclosure that possess a sufficiently basic, a sufficiently acidic, or both functional groups, can react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Alternatively, compounds that are inherently charged, such as those with a quaternary nitrogen, can form a salt with an appropriate counterion, e.g., chloride, a halide such as bromide, or fluoride, particularly bromide.

[0057] The compounds described herein may in some cases exist as diastereomers, enantiomers, or other stereoisomeric forms. The compounds presented herein may include all enantiomeric, diastereomeric, and/or epimeric forms as well as the appropriate mixtures thereof. Separation of stereoisomers may be performed by chromatography or by the forming diastereomeric and separation by chromatography, recrystallization, or any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley And Sons, Inc., 1981, herein incorporated by reference for this disclosure). Stereoisomers may also be obtained by stereoselective synthesis.

[0058] As used herein, the “pharmaceutically acceptable salt” can refer to those salts which are suitable for use in contact with the tissues of subjects without e.g. undue toxicity, irritation or allergic response and are commensurate with e.g. a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts have been described elsewhere. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1- 19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Inorganic acids from which salts can be derived include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, but are not limited to, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, ethanesulfonic acid, acetic acid, propionic acid, p-toluenesulfonic acid, and methanesulfonic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as phosphoric acid, perchloric acid sulfuric acid, hydrochloric acid, and/or hydrobromic acid, or with organic acids such as maleic acid, malonic acid, tartaric acid, citric acid, acetic acid, oxalic acid, and/or succinic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, adipate, alginate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, nicotinate, nitrate, oleate, oxalate, lactobionate, lactate, laurate, lauryl sulfate, p- toluenesulfonate, undecanoate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, and valerate salts. In some cases, organic acids from which salts can be derived include, for example, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, cinnamic acid, mandelic acid, salicylic acid, methanesulfonic acid, acetic acid, propionic acid, ethanesulfonic acid, p-toluenesulfonic acid, and benzoic acid.

[0059] Pharmaceutically acceptable salts derived from appropriate bases may include N⁺(Ci- 4alkyl)⁴-salts, alkali metal, alkaline earth metal, and/or ammonium. Inorganic bases from which salts can be derived include, but are not limited to, calcium, magnesium, iron, zinc, copper, manganese, sodium, potassium, lithium, ammonium, aluminum, and/or the like. Organic bases from which salts can be derived include, but are not limited to, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, basic ion exchange resins, cyclic amines, and/or the like, examples include, but are not limited to, diethylamine, triethylamine, tripropylamine, isopropylamine, trimethylamine, and/or ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is sodium, calcium, ammonium, potassium, or magnesium salts. Representative alkali or alkaline earth metal salts may include potassium, calcium, magnesium, sodium, lithium, iron, zinc, copper, manganese, and aluminum. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as carboxylate, aryl sulfonate, sulfate, nitrate, lower alkyl sulfonate halide, hydroxide, and phosphate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and/or the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from sodium, calcium, ammonium, potassium, and/or magnesium salts. Bis salts (e.g., two counterions) and higher salts e.g., three or more counterions) are encompassed within the meaning of pharmaceutically acceptable salts. [0060] The “substituted” can refer to moieties having substituents replacing a hydrogen on one or more substitutable heteroatoms or carbons, e.g., NH, of the structure. It will be understood that “substitution” or “substituted with” may include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, ie., a compound which does not spontaneously undergo transformation such as by cyclization, elimination, rearrangement, etc. In certain embodiments, substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an imino, oxo, or thioxo group. As used herein, the “substituted” can be contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, carbocyclic and heterocyclic, branched and unbranched, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.

[0061] In some embodiments, substituents may include any substituents described herein, for example: halogen, nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazino (=N- NH2), -R^b-OR^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), -R^b-OC(O)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R^b-C(O)OR^a, -R^b-C(O)N(R^a)₂, -R^b-O-R^c-C(O)N(R ^a)₂, -R^b-N(R^a)C(O)OR^a, -R^b-N(R^a)C(O)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2), and -R^b-S(O)tN(R^a)2 (where t is 1 or 2); and alkenyl, aralkyl, aralkenyl, aralkynyl, alkynyl, aryl, cycloalkyl, alkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH₂), -R^b-0R^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, -R^b-OC(O)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R^b-C(O)OR^a, -R^b-C(O)N(R^a)₂, -R^b-0-R^c-C(0)N(R ^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)2 (where t is 1 or 2); wherein each R^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R^a, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-OH), hydrazine (=N-

NH₂), -R^b-OR^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, -R^b-OC(O)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R ^b-C(O)OR^a, -R^b-C(O)N(R^a)₂, -R^b-O-R^c-C(O)N(R^a)₂, -R^b-N(R^a)C(O)OR^a, -R^b-N(R^a)C(O)R^a, -R^b- N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each R^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0062] In some embodiments, substituents can include any substituents described herein, for example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO₂), imino (=N-H), oximo (=N-0H), hydrazino (=N-NH₂), -R^b-OR^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, -R^b-OC(O)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R^b-C(O)OR^a, -R^b-C(O)N(R^a)₂, -R^b-O-R^c-C(O)N(R ^a)₂, -R^b-N(R^a)C(O)OR^a, -R^b-N(R^a)C(O)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2), and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and alkyl, alkenyl, and alkynyl each of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (- CN), nitro (-NO₂), imino (=N-H), oximo (=N-0H), hydrazine (=N-

NH₂), -R^b-0R^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R^b-C(O)OR^a, -R^b-C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N( R^a)C(O)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R^a, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO₂), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH₂), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R ^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(0)_tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each R^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0063] In some embodiments, substituents can include any substituents described herein, for example: halogen, haloalkyl, oxo (=0), hydroxy, thioxo (=S), cyano (-CN), nitro (-NO₂), imino (=N-H), oximo (=N-0H), hydrazino (=N-NH₂), -R^b-0R^a, -R^b-OC(O)-R^a, -R^b-OC(O)-OR^a, -R^b-OC(O)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(O)R^a, -R^b-C(O)OR^a, -R^b-C(O)N(R^a)₂, -R ^b-O-R^c-C(O)N(R^a)₂, -R^b-N(R^a)C(O)OR^a, -R^b-N(R^a)C(O)R^a, -R^b-N(R^a)S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2), and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, wherein the alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl each of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-N0₂), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH₂), -R^b-0R^a, -R^b-0C(0)-R^a,

-R^b-0C(0)-0R^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R ^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(0)_tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R^a, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, hydoxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-N0₂), imino (=N-H), oximo (=N-0H), hydrazine (=N-

NH₂), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R ^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b- N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each R^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0064] In some embodiments, substituents can include any substituents described herein, for example: halogen, hydroxy, fluoroalkyl, oxo (=0), cyano (-CN), nitro (-

NO₂), -R^b-0R^a, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, and -R^b-N(R^a)C(0)R^a; and alkyl, aryl, cycloalkyl, heterocycloalkyl, and heteroaryl, each of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), hydroxy, thioxo (=S), cyano (-CN), nitro (-N0₂), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH₂), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a,

-R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R ^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)₂ (where t is 1 or 2); and wherein each R^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R^a, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazine (=N-

NH₂), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R ^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b- N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(0)tN(R^a)2 (where t is 1 or 2); and wherein each R^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each R^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0065] In some embodiments, substituents can include any substituents described herein, for example: alkyl, halo, fluoroalkyl, oxo (=0), hydroxy, cyano (-CN), -R^b-0R^a, -R^b-N(R^a)2, -R^b-C(0)R^a, and -R^b-C(0)0R^a, wherein the alkyl may be optionally substituted by alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (- CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH2), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a, -R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b- C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(0)_tR^a (where t is 1 or 2), -R^b-S(O)tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)2 (where t is 1 or 2); and wherein each R^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R^a, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazine (=N-NH₂), -R^b-0R^a, -R^b-0C(0)-R^a, -R^b-0C(0)-0R^a,

-R^b-0C(0)-N(R^a)₂, -R^b-N(R^a)₂, -R^b-C(0)R^a, -R^b-C(0)0R^a, -R^b-C(0)N(R^a)₂, -R^b-0-R^c-C(0)N(R ^a)₂, -R^b-N(R^a)C(0)0R^a, -R^b-N(R^a)C(0)R^a, -R^b-N(R^a)S(O)tR^a (where t is 1 or 2), -R^b-S(O)_tR^a (where t is 1 or 2), -R^b-S(O)tOR^a (where t is 1 or 2) and -R^b-S(O)tN(R^a)2 (where t is 1 or 2); and wherein each R^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each R^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0066] In addition, if a compound of the present disclosure is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if a product is a free base, an acid addition salt, particularly a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds.

[0067] As used herein, the “solvate” can refer to compounds that further include a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. The solvate can be of a disclosed compound or a pharmaceutically acceptable salt thereof. Where the solvent is water, the solvate is a “hydrate”. Pharmaceutically acceptable solvates and hydrates are complexes that, for example, can include 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules. In some embodiments, the solvate can be a channel solvate. It will be understood that the “compound” as used herein encompasses the compound and solvates of the compound, as well as mixtures thereof.

[0068] The “analyte” or “analytes,” as used herein, can refer to a molecule whose presence or absence is measured or identified. An analyte can be a molecule for which a detectable probe or assay exists or can be produced. For example, an analyte can be a macromolecule, such as, for example, a nucleic acid, a polypeptide, a carbohydrate, a small organic, an inorganic compound, or an element, for example, gold, iron, or lead. An analyte can be part of a sample that contains other components, or can be the sole or the major component of the sample. An analyte can be a component of a whole cell or tissue, a cell or tissue extract, a fractionated lysate thereof or a substantially purified molecule. In some embodiments, the target analyte is a polypeptide.

[0069] Thes “polypeptide” and “peptide” generally to refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non- naturally occurring amino acid (i.e., amino acid analogue). The polymer can be linear or branched and can include modified amino acids, and/or may be interrupted by non-amino acids. Polypeptides can occur as single chains or associated chains. The polymer may include a plurality of amino acids and may have a secondary and tertiary structure (i.e., protein). In some examples, the polymer comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1000, at least about 10,000, or more amino acids.

[0070] The “amino acid,” as used herein, can refer to a naturally occurring or non-naturally occurring amino acid (amino acid analogue). The non-naturally occurring amino acid may be a synthesized amino acid. As used herein, the “amino acid sequence,” “peptide sequence,” and “polypeptide sequence,” as used herein, can refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide (amide) bond or an analog of a peptide bond. The peptide includes oligomers and polymers of amino acids or amino acid analogs. The amino acids of the peptide may be L-amino acids or D-amino acids. A peptide, polypeptide, or protein may be synthetic, recombinant, or naturally occurring. A synthetic peptide may be a peptide that is produced by artificial approaches in vitro.

[0071] Thes “amino acid sequence,” “peptide sequence,” and “polypeptide sequence,” as used herein, generally refer to a sequence of at least two amino acids or amino acid analogs that are covalently linked (e.g., by a peptide (amide) bond or an analog of a peptide bond). A peptide sequence may refer to a complete sequence or a portion of a sequence. For example, a peptide sequence may contain gaps, positions with unknown identities, or positions that can accommodate distinct species.

[0072] As used herein, the “side chain” or “R-group” can refer to structures attached to an amino acid alpha carbon (attaching the amine and carboxylic acid groups of the amino acid) that render uniqueness to each type of amino acid. R groups have a variety of shapes, sizes, charges, and reactivities, such as charged polar side chains, either positively or negatively charged, such as lysine (+), arginine (+), histidine (+), aspartate (-), and glutamate (-); amino acids can also be basic, such as lysine, or acidic, such as glutamic acid; uncharged polar side chains have hydroxyl, amide, or thiol groups, such as cysteine having a chemically reactive side chain, i.e., a thiol group that can form bonds with another cysteine, serine (Ser) and threonine (Thr), that have hydroxylic R side chains of different sizes; asparagine (Asn), glutamine (Gin), and tyrosine (Tyr); non-polar hydrophobic amino acid side chains include the amino acid glycine, alanine, valine, leucine, and isoleucine having aliphatic hydrocarbon side chains ranging in size from a methyl group for alanine to isomeric butyl groups for leucine and isoleucine; methionine (Met) has a thiol ether side chain; proline (Pro) has a cyclic pyrrolidine side group. Phenylalanine (with its phenyl moiety) (Phe) and tryptophan (Trp) (with its indole group) contain aromatic side chains, which are characterized by bulk as well as lack of polarity.

[0073] The “cleavable unit,” as used herein, can refer to a molecule that can be split into at least two molecules. Non-limiting examples of cleavage reagents and conditions to split a cleavable unit include: enzymes, nucleophilic or basic reagents, reducing agents, photoirradiation, electrophilic or acidic reagents, organometallic or metal reagents, and oxidizing reagents.

[0074] The “sample,” as used herein, can refer to a sample containing or suspected of containing a polypeptide. For example, a sample can be a biological sample containing one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In some examples, the sample is obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In some examples, the sample can include circulating tumor cells. In some examples, the sample is an environmental sample (e.g., soil, waste, ambient air), industrial sample (e.g., samples from any industrial processes), and food samples (e.g., dairy products, vegetable products, and meat products). The sample may be processed prior to loading into a microfluidic device. For example, the sample may be processed to purify the polypeptides and/or to include reagents.

[0075] As used herein, sequencing of peptides “at the single molecule level” can refer to amino acid sequence information obtained from individual (i.e., single) peptide molecules in a mixture of diverse peptide molecules. The amino acid sequence information may be obtained from an entirety of an individual peptide molecule or one or more portion of the individual peptide molecule, such as a contiguous amino acid sequence of at least a portion of the individual peptide molecule. Alternatively, partial amino acid sequence information may be obtained, which may allow for identification of the peptide or protein. Partial amino acid sequence information, including for example, the pattern of a specific amino acid residue (i.e., lysine) within individual peptide molecules, may be sufficient to uniquely identify an individual peptide molecule. For example, a pattern of amino acids may comprise a plurality of identified positions (e.g., identified as a particular amino acid type, such as lysine, or identified as a particular set of amino acids, such as the set of carboxylate side chain-containing amino acids), and a plurality of unidentified positions. The sequence of identified positions may be searched against a known proteome of a given organism to identify the individual peptide molecule. In some examples, sequencing of a peptide at the single molecule level may identify a pattern of a certain type of amino acid (e.g., lysine) in an individual peptide molecule. Such information may be used to identify a macromolecule (e.g., protein) from which the peptide was derived. This may advantageously preclude the need to identify all amino acids of the peptide.

[0076] As used herein, the “Edman degradation” can refer to methods comprising chemical removal of amino acids from peptides or proteins. In some cases, Edman degradation denotes terminal (e.g., N- or C-terminal) amino acid removal. In specific cases, Edman degradation refers to N-terminal amino acid removal through isothiocyanate (e.g., phenyl isothiocyanate) coupling and cyclization with the terminal amine group of an N-terminal residue, such that the N-terminal amino acid is removed from a peptide. In some cases, Edman degradation refers to N-terminal amino acid removal through use of any of the Edman reagents described herein in place of isothiocyanate (e.g. a compound of Formula I, a compound of Formula II, or any combination thereof). In some cases, Edman degradation broadly encompasses N-terminal amino acid functionalizations leading to N-terminal amino acid removal. In some cases, Edman degradation encompasses C-terminal amino acid removal. In some cases, Edman degradation comprises terminal amino acid functionalization (e.g., N-terminal amino acid isothiocyanate functionalization) followed by enzymatic removal (e.g., by an ‘Edmanase’ with specificity for chemically derivatized N-terminal amino acids).

[0077] As used herein, the “single molecule sensitivity” can refer to the ability to acquire data (including, for example, amino acid sequence information) from individual peptide molecules in a mixture of diverse peptide molecules. In one non-limiting example, the mixture of diverse peptide molecules may be immobilized on a solid surface (including, for example, a glass slide, or a glass slide whose surface has been chemically modified). This may include the ability to simultaneously record the fluorescent intensity of multiple individual (i.e., single) peptide molecules distributed across the glass surface. Optical devices are commercially available that can be applied in this manner. For example, a conventional microscope equipped with total internal reflection illumination and an intensified charge-couple device (CCD) detector is available. Imaging with a high sensitivity CCD camera allows the instrument to simultaneously record the fluorescent intensity of multiple individual (i.e., single) peptide molecules distributed across a surface. Image collection may be performed using an image splitter that directs light through two band pass filters (one suitable for each fluorescent molecule) to be recorded as two side-by-side images on the CCD surface. Using a motorized microscope stage with automated focus control to image multiple stage positions in the flow cell may allow millions of individual single peptides (or more) to be sequenced in one experiment. [0078] As used herein, the “array” can refer to a population of sites. Such populations of sites can be differentiated from one another according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single polypeptide having a particular sequence or a site can include several polypeptides having the same sequence. The sites of an array can be different features located on the same substrate. Such features may include, without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be separate substrates each bearing at least one molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Such different molecules may have the same or different sequences. An array may include one or more wells, and an well of the one or more wells may have one or more beads. As an alternative, the array may be a planar surface having, for example, a molecule immobilized thereon, or, as another example, one or more beads immobilized thereon.

[0079] As used herein, the term “label” can refer to a molecular or macromolecular construct that can couple to a reactive group, such as an amino acid side chain, C-terminal carboxylate, or N-terminal amine. The label may comprise at least one reactive group (e.g., a first reactive group and a second reactive group). The at least one reactive group may be configured to couple to a polypeptide. The at least one reactive group may be configured to couple to a support. The at least one reactive group may be coupled to or configured to couple to a detectable moiety. A label may provide a measurable signal.

[0080] As used herein, the “polymer matrix” can refer to a continuous phase material that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid or gaseous species. For example, the ‘polymer matrix’ may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. [0081] Peptide sequence information may be obtained from a polypeptide molecule or from one or more portions of the polypeptide molecule. Peptide sequencing may provide complete or partial amino acid sequence information for a peptide sequence or a portion of a peptide sequence. At least a portion of the peptide sequence may be determined at the single molecule level. In some cases, partial amino acid sequence information, including for example, the relative positions of a specific type of amino acid (e.g., lysine) within a peptide or portion of a peptide, may be sufficient to uniquely identify an individual peptide molecule. For example, a pattern of amino acids, such as, for example, X-X-X-Lys-X-X-X-X-Lys-X-Lys, which indicates the distribution of lysine molecules within an individual peptide molecule, may be searched against a known proteome of a given organism to identify the individual peptide molecule. Such information may be used to identify a macromolecule (e.g., protein) from which the peptide was derived, and may preclude the need to identify all amino acids of the peptide. Degradation Agent

[0082] Provided herein are methods, compositions, systems, and/or kits for degrading one or more amino acids from a sample (e.g., polypeptide). Compared to Hamada degradation methods may be used to facilitate terminal amino acid removal comprising a terminal amino acid derivatization step or operation and a subsequent cleavage step or operation, but required the use of hazardous chemicals like hydrazine (FIG. 2). Additionally, the use of a crosslinker may lead to unwanted crosslinking reactions with nearby nucleophiles, preventing further sequencing of the peptide fragment.

[0083] Responsive to the present needs for faster, chemically less intensive, and higher efficiency amino acid removal, the present disclosure provides a degradation agent capable not only of cleaving a terminal amino acid (e.g., a N-terminal amino acid or C-terminal amino acid) or an internal amino acid under basic aqueous conditions but also of readily guanidinylating in the same buffer without the need for dangerous chemicals or reagents. Furthermore, the degradation agent may be controlled between the guanidinylation and cleavage step or operations to prevent continuous, uninhibited removal of N-terminal amino acids, which may otherwise result in complete digestion of the peptide. In some cases, photocaging the degradation agent may allow for a "trigger-and-release" cleavage mechanism, maintaining a singular aqueous buffer without additives (FIG. 3). In addition, using a photocage can provide spatial and temporal control of cleavage, giving users greater flexibility in investigating peptides.

[0084] In some aspects, the present disclosure provides a degradation agent for modifying and subsequently degrading a terminal amino acid e.g., N-terminal amino acid or C-terminal amino acid) of a polypeptide.

[0085] In some cases, the degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or

3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety.

[0086] LG, a leaving group, can be an atom or a group of atoms that detaches from the main or residual part of a substrate during a reaction or elementary step of a reaction. In the present disclosure, a leaving group may detach from the degradation agent upon reaction with a terminal amino acid (e.g., N-terminal amino acid or C-terminal amino acid) of a polypeptide. In some cases, LG may be perfluoroalkyl sulfonyl (e.g., triflyl), thioester, sulfonate, diazole, triazole, aminotriflate, benzazole, sulfonamides, sulfonimide, sulfonic ester, sulfoxide, sulfones, phosphates, or phosphoesters. In some cases, LG is -SR³ or -SCL', wherein R³ is Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted.

[0087] In some cases, R¹ is hydrogen. In some cases, R¹ is C1-6 alkyl. In some cases, R¹ is C2-6 alkenyl. In some cases, R¹ is C2-6 alkynyl. In some cases, R¹ is C3-10 carbocyclyl. In some cases, R¹ is 3- to 10-membered heterocyclyl. In some cases, the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted. In some cases, the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted with one or more R^u.

[0088] In some cases, R² is hydrogen. In some cases, R² is C1-6 alkyl. In some cases, R² is C2-6 alkenyl. In some cases, R² is C2-6 alkynyl. In some cases, R² is C3-10 carbocyclyl. In some cases, R² is 3- to 10-membered heterocyclyl. In some cases, the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted. In some cases, the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted with one or more R^u.

[0089] In some cases, both R¹ and R² are hydrogen.

[0090] In some cases, each R^u is independently selected from oxo, halogen, -CN, -NO2, -

OH, -NH2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, C2-6 alkenyl, C2-6 alkynyl, C3-12 carbocyclyl, 3- to 12-membered heterocyclyl, Ce-io aryl (e.g., phenyl or naphthyl), 5- to 10-membered heteroaryl e.g., heteroaryl comprising one or two 5- or 6-membered rings and 1-5 heteroatoms selected from N, O, and S), -SR^b, -S(=O)R^a, -S(=O)₂R^a, -S(=O)₂OR^b, -S(=O)₂NR^cR^d, - NR^cS(=O)₂R^a, -NR^cS(=O)R^a, -NR^cS(=O)₂OR^b, -NR^cS(=O)₂NR^cR^d, -NR^bC(=O)NR^cR^d, - NR^bC(=O)R^a, -NR^bC(=O)OR^b, -OS(=O)₂R^a, -OS(=O)₂OR^b, -OS(=O)₂NR^cR^d, -OC(=O)R^a, - OC(=O)OR^b, -OC(=O)NR^cR^d, -C(=O)R^a, -C(=O)OR^b, and -C(=O)NR^cR^d; wherein the alkyl, alkoxy, alkylamino, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from oxo, halogen, -CN, -NO2, -OH, -NH2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, and 3- to 6- membered heterocyclyl. [0091] In some cases, each R^a is independently Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-12 carbocyclyl, 3- to 12-membered heterocyclyl, Ce-io aryl, or 5- to 10-membered heteroaryl. In some cases, each R^a is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, 3- to 6-membered heterocyclyl, Ce aryl, or 5- to 6-membered heteroaryl. In some cases, each R^a is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, or 3- to 6-membered heterocyclyl. In some cases, each R^a is independently C1-6 alkyl, C3-6 carbocyclyl, or 3- to 6- membered heterocyclyl.

[0092] In some cases, each R^b is independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-12 carbocyclyl, 3- to 12-membered heterocyclyl, Ce-io aryl, or 5- to 10-membered heteroaryl. In some cases, each R^b is independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, 3- to 6-membered heterocyclyl, Ce aryl, or 5- to 6-membered heteroaryl. In some cases, each R^b is independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, or 3- to 6-membered heterocyclyl. In some cases, each R^b is independently hydrogen, C1-6 alkyl, C3-6 carbocyclyl, or 3- to 6-membered heterocyclyl.

[0093] In some cases, each R^c and each R^d is independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-12 carbocyclyl, 3- to 12-membered heterocyclyl, Ce-io aryl, or 5- to 10- membered heteroaryl. In some cases, each R^c and each R^d is independently hydrogen, C1-6 alkyl, C3-6 carbocyclyl, or 3- to 6-membered heterocyclyl. In some cases, R^c and R^d, together with the nitrogen atom to which they are attached, form 3- to 12-membered heterocyclyl.

[0094] In some cases, R^a, R^b, R^c, and R^d is independently and optionally substituted with one or more R^z. In some cases, R^z is independently oxo, halogen, -CN, -NO2, -OH, -NH2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, or 3- to 6- membered heterocyclyl.

[0095] In some cases, PC is a photo-cleavable moiety. Photo-cleavable moi eties, (e.g., photo-releasable or photo-activatable moieties), may be protecting groups that provide spatial and temporal control over the release of various chemicals. Such photo-cleavable moieties may be removable upon subjecting to a light source having certain wavelength, but are otherwise stable under other various conditions (e.g., pH, temperature, oxidation, etc.).

[0096] In some cases, the photo-cleavable moiety comprises one or more aromatic groups. In some cases, the photo-cleavable moiety comprises one or more substituted or unsubstituted Ce-i4 aryl or substituted or unsubstituted 5- to 14-membered heteroaryl. In some cases, the photo-cleavable moiety comprises one or more substituted Ce-14 aryl or substituted 5- to 14- membered heteroaryl. In some cases, the photo-cleavable moiety comprises a substituted phenyl. In some cases, the photo-cleavable moiety comprises a substituted pyridinyl. The substituents on the one or more aromatic group may be one or more substituents selected from oxo, halogen, - CN, -NO2, -OH, -NH2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, C2-6 alkenyl, C2-6 alkynyl, C3-12 carbocyclyl, 3- to 12-membered heterocyclyl, Ce-io aryl (e.g., phenyl or naphthyl), 5- to 10- membered heteroaryl (e.g., heteroaryl comprising one or two 5- or 6-membered rings and 1-5 heteroatoms selected from N, O, and S), -SR^b, -S(=O)R^a, -S(=O)2R^a, -S(=O)2OR^b, - S(=O)₂NR^cR^d, -NR^cS(=O)₂R^a, -NR^cS(=O)R^a, -NR^cS(=O)₂OR^b, -NR^cS(=O)₂NR^cR^d, - NR^bC(=O)NR^cR^d, -NR^bC(=O)R^a, -NR^bC(=O)OR^b, -OS(=O)₂R^a, -OS(=O)₂OR^b, - OS(=O)₂NR^cR^d, -OC(=O)R^a, -OC(=O)OR^b, -OC(=O)NR^cR^d, -C(=O)R^a, -C(=O)OR^b, and - C(=O)NR^cR^d; wherein the alkyl, alkoxy, alkylamino, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from oxo, halogen, -CN, -NO2, -OH, -NH2, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, C2-6 alkenyl, C2-6 alkynyl, C3-6 carbocyclyl, and 3- to 6-membered heterocyclyl.

[0097] The reagent may also be configured to couple to a terminus of the polypeptide. In some cases, the reagent is configured to couple to the N-terminus of the polypeptide. In some cases, the reagent is configured to couple to the C-terminus of the polypeptide. FIG. 17 provides a reaction scheme for N-terminal amino acid removal with a representative degradation agent of a structure of Formula I, comprising a carboxamidine group. As shown in the reaction scheme of FIG. 17, a terminal amine of a polypeptide of Formula II may react with the degradation agent, thereby forming a first modified polypeptide comprising a modified N-terminus comprising a guanidine group (Formula III). The guanidine group can comprise a photo-cleavable moiety. The first modified polypeptide may be stable until subjected to certain condition. In some cases, the condition may be a light source of certain wavelength, which removes the photo-cleavable moiety (Y), thereby forming an intermediate of Formula IV comprising an N-terminus modified with a guanidine group without a photo-cleavable moiety. The intermediate of Formula IV may undergo cyclization reaction to form a cyclic fragment of Formula V and a second modified peptide. The cyclic fragment of Formula V may comprise a residue of the N-terminal amino acid of the polypeptide.

[0098] The reagent may also be configured to couple to a terminus of the polypeptide. In some cases, the reagent may be configured to couple to the N-terminus of the polypeptide. FIG. 18 provides a reaction scheme for N-terminal amino acid removal with a representative degradation agent of a structure of Formula I’, comprising a carboxamidine group. As shown in the reaction scheme of FIG. 18, a terminal amine of a polypeptide of Formula II may react with the degradation agent, thereby forming a first modified polypeptide comprising a modified N- terminus comprising a guanidine group (Formula III’). The guanidine group can comprise a photo-cleavable moiety. The first modified polypeptide may be stable until subjected to certain condition. For example, in some cases, the first modified polypeptide may be stable for at least 10 minutes, at least 20 minutes, at least 40 minutes, at least 60 minutes, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 4 days, at least 6 days, at least 7 days, at least 2 weeks, at least 4 weeks, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, or more until subjected to certain conditions. In some cases, the first modified polypeptide may be stable for at most 10 minutes, at most 20 minutes, at most 40 minutes, at most 60 minutes, at most 2 hours, at most 4 hours, at most 6 hours, at most 8 hours, at most 10 hours, at most 15 hours, at most 20 hours, at most 24 hours, at most 2 days, at most 4 days, at most 6 days, at most 7 days, at most 2 weeks, at most 4 weeks, at most 2 months, at most 4 months, at most 6 months, at most 8 months, at most 10 months, at most 12 months, or less until subjected to certain conditions. In some cases, the condition is a light source of certain wavelength, which removes the photo-cleavable moiety (Y), thereby forming an intermediate of Formula IV’ comprising an N-terminus modified with a guanidine group without a photo- cleavable moiety. The intermediate of Formula IV’ may undergo cyclization reaction to form a cyclic fragment of Formula V’ and a second modified peptide. The cyclic fragment of Formula V’ may comprise a residue of the N-terminal amino acid of the polypeptide. In some cases, representative photo-cleavable moi eties may include, but not limited to, the ones in Table 1.

Table 1: Exemplary photo-cleavable moieties

X and R represent a parent molecule that is photocaged by the photo-cleavable moiety.

[0099] In some cases, the photo-cleavable moiety may comprise a nitro-substituted benzyl group. In some cases, the photo-cleavable moiety is

[00100] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a certain wavelength.

[00101] A light source having a certain wavelength provides photons with sufficient energy, which upon absorption, enables the photo reaction and leads to the photo cleavage. In general, the photo-cleavable moieties may have strong absorption at wavelengths of at least about 200 nm, where irradiation is less likely to be absorbed by (and possibly cause damage to) the biological entity (e.g., structures of polypeptide such as primary or secondary structures). Moreover, the photoreaction can be clean and can occur with a high quantum yield or efficiency for release.

[00102] In some cases, the photo-cleavable moiety may be cleavable when subjected to a light source having a wavelength of from about 200 nm to about 750 nm. In some cases, the photo-cleavable moiety may be cleavable when subjected to a light source having a wavelength of from about 200 nm to about 250 nm, from about 200 nm to about 300 nm, from about 200 nm to about 350 nm, from about 200 nm to about 400 nm, from about 200 nm to about 450 nm, from about 200 nm to about 500 nm, from about 200 nm to about 550 nm, from about 200 nm to about 600 nm, from about 200 nm to about 650 nm, from about 200 nm to about 700 nm, from about 200 nm to about 750 nm, from about 250 nm to about 300 nm, from about 250 nm to about 350 nm, from about 250 nm to about 400 nm, from about 250 nm to about 450 nm, from about 250 nm to about 500 nm, from about 250 nm to about 550 nm, from about 250 nm to about 600 nm, from about 250 nm to about 650 nm, from about 250 nm to about 700 nm, from about 250 nm to about 750 nm, from about 300 nm to about 350 nm, from about 300 nm to about 400 nm, from about 300 nm to about 450 nm, from about 300 nm to about 500 nm, from about 300 nm to about 550 nm, from about 300 nm to about 600 nm, from about 300 nm to about 650 nm, from about 300 nm to about 700 nm, from about 300 nm to about 750 nm, from about 350 nm to about 400 nm, from about 350 nm to about 450 nm, from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 350 nm to about 650 nm, from about 350 nm to about 700 nm, from about 350 nm to about 750 nm, from about 400 nm to about 450 nm, from about 400 nm to about 500 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 400 nm to about 700 nm, from about 400 nm to about 750 nm, from about 450 nm to about 500 nm, from about 450 nm to about 550 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 450 nm to about 750 nm, from about 500 nm to about 550 nm, from about 500 nm to about 600 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 600 nm, from about 550 nm to about 650 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 600 nm to about 650 nm, from about 600 nm to about 700 nm, from about 600 nm to about 750 nm, from about 650 nm to about 700 nm, from about 650 nm to about 750 nm, or about 700 nm to about 750 nm. In some cases, the photo-cleavable moiety may be cleavable when subjected to a light source having a wavelength of at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, or more. In some cases, the photo-cleavable moiety may be cleavable when subjected to a light source having a wavelength of at most about 250 nm, at most about 300 nm, at most about 350 nm, at most about 400 nm, at most about 450 nm, at most about 500 nm, at most about 550 nm, at most about 600 nm, at most about 650 nm, at most about 700 nm, at most about 750 nm or less.

[00103] In some cases, the photo-cleavable moiety may be cleavable when subjected to a light source having a wavelength of about 300 nm to about 500 nm, about 300 nm to about 480 nm, about 300 nm to about 460 nm, about 300 nm to about 440 nm, about 300 nm to about 420 nm, about 300 nm to about 400 nm, about 300 nm to about 380 nm, about 300 nm to about 360 nm, about 300 nm to about 340 nm, about 300 nm to about 320 nm, about 320 nm to about 500 nm, about 320 nm to about 480 nm, about 320 nm to about 460 nm, about 320 nm to about 440 nm, about 320 nm to about 420 nm, about 320 nm to about 400 nm, about 320 nm to about 380 nm, about 320 nm to about 360 nm, about 320 nm to about 340 nm, about 340 nm to about 500 nm, about 340 nm to about 480 nm, about 340 nm to about 460 nm, about 340 nm to about 440 nm, about 340 nm to about 420 nm, about 340 nm to about 400 nm, about 340 nm to about 380 nm, or about 340 nm to about 360 nm.

[00104] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of about 300 nm to about 500 nm or about 300 nm to about 400 nm. [00105] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of greater than about 300 nm, greater than about 310 nm, greater than about 320 nm, greater than about 330 nm, greater than about 340 nm, greater than about 350 nm, greater than about 360 nm, greater than about 370 nm, greater than about 380 nm, greater than about 390 nm, greater than about 400 nm, greater than about 410 nm, greater than about 420 nm, greater than about 430 nm, greater than about 440 nm, greater than about 450 nm, greater than about 460 nm, greater than about 470 nm, greater than about 480 nm, greater than about 490 nm, or greater than about 500 nm, or more.

[00106] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of greater than about 400 nm.

[00107] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of less than about 310 nm, less than about 320 nm, less than about 330 nm, less than about 340 nm, less than about 350 nm, less than about 360 nm, less than about

370 nm, less than about 380 nm, less than about 390 nm, less than about 400 nm, less than about

410 nm, less than about 420 nm, less than about 430 nm, less than about 440 nm, less than about 450 nm, less than about 460 nm, less than about 470 nm, less than about 480 nm, less than about 490 nm, or less than about 500 nm, or less.

[00108] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of less than about 400 nm.

[00109] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of about 300 nm, about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, or about 400 nm.

[00110] In some cases, the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of about 365 nm.

Methods of Modifying and Degrading a Terminal Amino Acid of a Polypeptide

[00111] In some aspects, the present disclosure provides methods for modifying a terminal amino acid. The method can comprise modifying a terminus (e.g., N-terminal amino acid or C- terminal amino acid) of a polypeptide. The terminus of the polypeptide can be contacted with a degradation agent comprising a photo-cleavable moiety disclosed herein. A first modified polypeptide comprising the photo-cleavable moiety may be formed.

[00112] The degradation agent may be configured to couple to a terminal amino acid (e.g., the N-terminal amino acid). In some cases, the degradation agent is configured to couple to an amine of the polypeptide’s N-terminal amino acid. In some cases, the degradation agent can be configured to couple to non-secondary amines, such as primary amines. In some cases, the degradation agent can be engineered to couple to terminal amines (e.g., N-terminal amines) among proteinogenic amino acid types. The degradation agent can be engineered to attach to terminal amines of non-natural amino acid types or natural amino acid types. The amino acid types can comprise modified or unmodified amino acids. Examples can comprise chemically derivatized amino acids, proteinogenic amino acids, post-translationally modified amino acids, or any combination thereof. In some cases, the degradation agent is configured to couple to N- terminal amines.

[00113] In some other aspects, the method further comprises degrading the terminal amino acid. The method may comprise subjecting at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide. The method may simultaneously generates a fragment comprising a residue of the terminal amino acid of the polypeptide. In some cases, the second modified polypeptide comprises one less ammo acid than the first polypeptide.

[00114] Because the reaction requires two separate inputs for degrading polypeptide, the degradation agent as well as light and basic aqueous buffer for N-terminal coupling reagent cleavage, the number of terminal amino acids removed from the peptide can be controlled, such that one or more (e.g., one) terminal amino acid is removed each cycle.

[00115] The condition may comprise a light source. A light source provides photons with sufficient energy, which upon absorption, enables the photo reaction and leads to the photo cleavage. In some cases, the photo-cleavable moieties may have strong absorption at wavelengths well above 300 nm, where irradiation is less likely to be absorbed by (and possibly cause damage to) the biological entity. Moreover, the photoreaction may be clean and occur with a high quantum yield or efficiency for release.

[00116] In some cases, the light source has a wavelength of from about 200 nm to about 750 nm. In some cases, the light source has a wavelength of about 200 nm to about 250 nm, from about 200 nm to about 300 nm, from about 200 nm to about 350 nm, from about 200 nm to about 400 nm, from about 200 nm to about 450 nm, from about 200 nm to about 500 nm, from about 200 nm to about 550 nm, from about 200 nm to about 600 nm, from about 200 nm to about 650 nm, from about 200 nm to about 700 nm, from about 200 nm to about 750 nm, from about 250 nm to about 300 nm, from about 250 nm to about 350 nm, from about 250 nm to about 400 nm, from about 250 nm to about 450 nm, from about 250 nm to about 500 nm, from about 250 nm to about 550 nm, from about 250 nm to about 600 nm, from about 250 nm to about 650 nm, from about 250 nm to about 700 nm, from about 250 nm to about 750 nm, from about 300 nm to about 350 nm, from about 300 nm to about 400 nm, from about 300 nm to about 450 nm, from about 300 nm to about 500 nm, from about 300 nm to about 550 nm, from about 300 nm to about 600 nm, from about 300 nm to about 650 nm, from about 300 nm to about 700 nm, from about 300 nm to about 750 nm, from about 350 nm to about 400 nm, from about 350 nm to about 450 nm, from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 350 nm to about 650 nm, from about 350 nm to about 700 nm, from about 350 nm to about 750 nm, from about 400 nm to about 450 nm, from about 400 nm to about 500 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 400 nm to about 700 nm, from about 400 nm to about 750 nm, from about 450 nm to about 500 nm, from about 450 nm to about 550 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 450 nm to about 750 nm, from about 500 nm to about 550 nm, from about 500 nm to about 600 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 600 nm, from about 550 nm to about 650 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 600 nm to about 650 nm, from about 600 nm to about 700 nm, from about 600 nm to about 750 nm, from about 650 nm to about 700 nm, from about 650 nm to about 750 nm, or about 700 nm to about 750 nm. In some cases, the light source has a wavelength of at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm or more. In some cases, the light source has a wavelength of at most about 250 nm, at most about 300 nm, at most about 350 nm, at most about 400 nm, at most about 450 nm, at most about 500 nm, at most about 550 nm, at most about 600 nm, at most about 650 nm, at most about 700 nm, at most about 750 nm or less.

[00117] In some cases, the light source has a wavelength of about 300 nm to about 500 nm, 300 nm to about 480 nm, 300 nm to about 460 nm, 300 nm to about 440 nm, 300 nm to about 420 nm, 300 nm to about 400 nm, 300 nm to about 380 nm, 300 nm to about 360 nm, 300 nm to about 340 nm, 300 nm to about 320 nm, about 320 nm to about 500 nm, 320 nm to about 480 nm, 320 nm to about 460 nm, 320 nm to about 440 nm, 320 nm to about 420 nm, 320 nm to about 400 nm, 320 nm to about 380 nm, 320 nm to about 360 nm, 320 nm to about 340 nm, about 340 nm to about 500 nm, 340 nm to about 480 nm, 340 nm to about 460 nm, 340 nm to about 440 nm, 340 nm to about 420 nm, 340 nm to about 400 nm, 340 nm to about 380 nm, or 340 nm to about 360 nm.

[00118] In some cases, the light source has a wavelength of greater than about 300 nm, greater than about 310 nm, greater than about 320 nm, greater than about 330 nm, greater than about 340 nm, greater than about 350 nm, greater than about 360 nm, greater than about 370 nm, greater than about 380 nm, greater than about 390 nm, greater than about 400 nm, greater than about 410 nm, greater than about 420 nm, greater than about 430 nm, greater than about 440 nm, greater than about 450 nm, greater than about 460 nm, greater than about 470 nm, greater than about 480 nm, or greater than about 490 nm, greater than about 500 nm, or more.

[00119] In some cases, the light source has a wavelength of less than about 310 nm, less than about 320 nm, less than about 330 nm, less than about 340 nm, less than about 350 nm, less than about 360 nm, less than about 370 nm, less than about 380 nm, less than about 390 nm, less than about 400 nm, less than about 410 nm, less than about 420 nm, less than about 430 nm, less than about 440 nm, less than about 450 nm, less than about 460 nm, less than about 470 nm, less than about 480 nm, less than about 490 nm, or less than about 500 nm, or less.

[00120] In some cases, the light source has a wavelength of about 300 nm, about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, or about 400 nm. [00121] In some cases, the light source has a wavelength of about 365 nm.

[00122] The condition may further comprise a basic aqueous buffer. The basic aqueous buffer provides a basic condition that facilitates the deprotonation of the proton on the guanidinyl and the cyclization that leads to the removal of one or more terminal amino acids.

[00123] In some cases, the basic buffer has a pH value of about 8.0 to about 14.0, about 8.0 to about 13.5, about 8.0 to about 13.0, about 8.0 to about 12.5, about 8.0 to about 12.0, about 8.0 to about 11.5, about 8.0 to about 11.0, about 8.0 to about 10.5, about 8.0 to about 10.0, about 8.0 to about 9.5, about 8.0 to about 9.0, about 8.5 to about 14.0, about 8.5 to about 13.5, about 8.5 to about 13.0, about 8.5 to about 12.5, about 8.5 to about 12.0, about 8.5 to about 11.5, about 8.5 to about 11.0, about 8.5 to about 10.5, about 8.5 to about 10.0, about 8.5 to about 9.5, about 8.5 to about 9.0, about 9.0 to about 14.0, about 9.0 to about 13.5, about 9.0 to about 13.0, about 9.0 to about 12.5, about 9.0 to about 12.0, about 9.0 to about 11.5, about 9.0 to about 11.0, about 9.0 to about 10.5, about 9.0 to about 10.0, about 9.0 to about 9.5, about 9.5 to about 14.0, about 9.5 to about 13.5, about 9.5 to about 13.0, about 9.5 to about 12.5, about 9.5 to about 12.0, about 9.5 to about 11.5, about 9.5 to about 11.0, about 9.5 to about 10.5, about 9.5 to about 10.0, about 10.0 to about 14.0, about 10.0 to about 13.5, about 10.0 to about 13.0, about 10.0 to about 12.5, about 10.0 to about 12.0, about 10.0 to about 11.5, about 10.0 to about 11.0, about 10.0 to about 10.5, about 10.5 to about 14.0, about 10.5 to about 13.5, about 10.5 to about 13.0, about 10.5 to about 12.5, about 10.5 to about 12.0, about 10.5 to about 11.5, about 10.5 to about 11.0, about 11.0 to about 14.0, about 11.0 to about 13.5, about 11.0 to about 13.0, about 11.0 to about 12.5, about 11.0 to about 12.0, about 11.0 to about 11.5, about 11.5 to about 14.0, about 11.5 to about 13.5, about 11.5 to about 13.0, about 11.5 to about 12.5, or about 11.5 to about 12.0, about 12.0 to about 14.0, about 12.0 to about 13.5, about 12.0 to about 13.0, or about 12.0 to about 12.5.

[00124] In some cases, the basic aqueous buffer has a pH value of greater than about 8.0, greater than about 8.5, greater than about 9.0, greater than about 9.5, greater than about 10.0, greater than about 10.5, greater than about 11, greater than about 11.5, greater than about 12.0, greater than about 12.5, or greater than about 13.0, or more. [00125] In some cases, the basic aqueous buffer has a pH value of less than about 8.0, less than about 8.5, less than about 9.0, less than about 9.5, less than about 10.0, less than about 10.5, less than about 11, less than about 11.5, less than about 12.0, less than about 12.5, or less than about 13.0, or less.

[00126] In some cases, the basic aqueous buffer has a pH value of about 8.0, about 8.2, about

8.4, about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, about 10.2, about 10.4, about 10.6, about 10.8, about 11.0, about 11.2, about 11.4, about 11.6, about 11.8, about 12.0, about 12.2, about 12.4, about 12.6, about 12.8, about 13.0, about 13.2, about

13.4, about 13.6, about 13.8, or about 14.0.

[00127] In some cases, the basic aqueous buffer has a pH value of about 13.0.

[00128] The degradation proceeds by nucleophilic attack of the guanidinyl nitrogen on the carbonyl of the terminal amino acid, thereby forming a cyclic compound V as shown in FIG. 17. [00129] The cyclic compound may be completely generated in less than 1 min, less than 2 min, less than 3 min, less than 4 min, less than 5 min, less than 6 min, less than 7 min, less than 8 min, less than 9 min, less than 10 min, less than 12 min, less than 14 min, less than 16 min, less than 18 min, less than 20 min, less than 22 min, less than 24 min, less than 26 min, less than 28 min, less than 30 min, less than 35 min, less than 40 min, less than 45 min, less than 50 min, less than 55 min, less than 60 min, less than 65 min, less than 70 min, less than 75 min, less than 80 min, less than 85 min, less than 90 min, less than 95 min, less than 100 min, less than 105 min, less than 110 min, less than 115 min, less than 120 min, less than 130 min, less than 140 min, less than 150 min, less than 160 min, less than 170 min, less than 180 min, less than 190 min, less than 200 min, less than 210 min, less than 220 min, less than 230 min, less than 240 min, less than 270 min, less than 300 min, less than 330 min, less than 360 min, less than 390 min, less than 420 min, less than 450 min, less than 480 min, less than 540 min, less than 600 min, less than 660 min, less than 720 min, less than 780 min, less than 840 min, or less than 900 min, following contacting the degradation agent with the polypeptide.

[00130] In some cases, the method disclosed herein further comprises, prior to (a), providing the polypeptide having a probe coupled to an amino acid of the polypeptide.

[00131] In some cases, the polypeptide is labelled with a bar code.

[00132] In some cases, the probe exhibits different spectral properties when coupled to different amino acids.

[00133] In some cases, the probe comprises a dipyrromethene-BF2 derivative.

[00134] In some cases, the method disclosed herein further comprises, detecting a signal or signal change from the probe to identify at least a portion of a sequence of the polypeptide. [00135] Compounds herein can include all stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.

[00136] Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.

[00137] Non-limiting examples of alkyl and alkylene groups include straight, branched, and cyclic alkyl and alkylene groups. An alkyl or alkylene group can be, for example, a Ci, C2, C3, C₄, C₅, C₆, C₇, C₈, C₉, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, c₂₅, C26, C27, C28, C29, C30, C31, C32, C33, C34, c₃₅, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C₄6, C47, C48, C49, or C50 group that is substituted or unsubstituted.

[00138] Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[00139] Branched alkyl groups include any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl.

[00140] Non-limiting examples of substituted alkyl groups includes hydroxymethyl, chloromethyl, trifluoromethyl, aminomethyl, 1 -chloroethyl, 2 -hydroxy ethyl, 1,2-difluoroethyl, and 3 -carboxypropyl.

[00141] Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also include fused-, bridged-, and spiro-bicycles and higher fused-, bridged-, and spiro-systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups. Non-limiting examples of cyclic alkyl groups include cyclopropyl, 2-methyl-cycloprop-l-yl, cycloprop-2-en-l-yl, cyclobutyl, 2,3-dihydroxycyclobut-l-yl, cyclobut-2-en-l-yl, cyclopentyl, cyclopent-2-en-l-yl, cyclopenta-2,4-dien-l-yl, cyclohexyl, cyclohex-2-en-l-yl, cycloheptyl, cyclooctanyl, 2,5-dimethylcyclopent-l-yl, 3,5-dichlorocyclohex-l-yl, 4-hydroxycyclohex-l-yl, 3,3,5-trimethylcyclohex-l-yl, octahydropentalenyl, octahydro- 1/7-indenyl, 3a, 4, 5, 6, 7,7a- hexahydro-3Z7-inden-4-yl, decahydroazulenyl, bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, l,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl. [00142] Non-limiting examples of alkenyl and alkenylene groups include straight, branched, and cyclic alkenyl groups. The olefin or olefins of an alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. An alkenyl or alkenylene group can be, for example, a C2, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, c₂₅, C26, C27, C28, C29, C30, C31, C32, C33, C34, c₃₅, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkenyl and alkenylene groups include ethenyl, prop-l-en-l-yl, isopropenyl, but-l-en-4-yl; 2- chloroethenyl, 4-hydroxybuten-l-yl, 7-hydroxy-7-methyloct-4-en-2-yl, and 7-hydroxy-7- methyloct-3 , 5 -dien-2-yl .

[00143] Non-limiting examples of alkynyl or alkynylene groups include straight, branched, and cyclic alkynyl groups. The triple bond of an alkylnyl or alkynylene group can be internal or terminal. An alkylnyl or alkynylene group can be, for example, a C2, C3, C4, C5, Ce, C₇, Cs, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C₂0, C₂1, C₂2, C₂3, C₂4, C₂₅, C₂6, C₂7, C₂8, C₂9, C₃0, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of alkynyl or alkynylene groups include ethynyl, prop-2-yn-l-yl, prop-l-yn-l-yl, and 2-methyl-hex-4-yn-l-yl; 5-hydroxy- 5-methylhex-3-yn-l-yl, 6-hydroxy-6-methylhept-3-yn-2-yl, and 5-hydroxy-5-ethylhept-3-yn-l- yi.

[00144] A halo-alkyl group can be any alkyl group substituted with any number of halogen atoms, for example, fluorine, chlorine, bromine, and iodine atoms. A halo-alkenyl group can be any alkenyl group substituted with any number of halogen atoms. A halo-alkynyl group can be any alkynyl group substituted with any number of halogen atoms.

[00145] An alkoxy group can be, for example, an oxygen atom substituted with any alkyl, alkenyl, or alkynyl group. An ether or an ether group comprises an alkoxy group. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and isobutoxy.

[00146] An aryl group can be heterocyclic or non-heterocyclic. An aryl group can be monocyclic or polycyclic. An aryl group can be substituted with any number of substituents described herein, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. Non-limiting examples of aryl groups include phenyl, toluyl, naphthyl, pyrrolyl, pyridyl, imidazolyl, thiophenyl, and furyl. Non-limiting examples of substituted aryl groups include 3,4- dimethylphenyl, 4-/c/7-butyl phenyl, 4-cyclopropylphenyl, 4-diethylaminophenyl, 4- (trifluoromethyl)phenyl, 4-(difluoromethoxy)-phenyl, 4-(trifluoromethoxy)phenyl, 3- chlorophenyl, 4-chlorophenyl, 3, 4-di chlorophenyl, 2-fluorophenyl, 2-chlorophenyl, 2- iodophenyl, 3 -iodophenyl, 4-iodophenyl, 2-m ethylphenyl, 3 -fluorophenyl, 3 -methylphenyl, 3- methoxyphenyl, 4-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2,3-difluorophenyl, 3,4- difluorophenyl, 3,5-difluorophenyl, 2,3 -dichlorophenyl, 3, 4-di chlorophenyl, 3,5-dichlorophenyl, 2-hydroxyphenyl, 3 -hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl, 3 -methoxyphenyl, 4- methoxyphenyl, 2,3 -dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 2,4- difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 2,3,4-trifluorophenyl, 2,3,5- trifluorophenyl, 2,3,6-trifluorophenyl, 2,4,5-trifluorophenyl, 2,4,6-trifluorophenyl, 2,4- di chlorophenyl, 2,5-dichlorophenyl, 2,6-dichlorophenyl, 3, 4-di chlorophenyl, 2,3,4- tri chlorophenyl, 2,3,5-trichlorophenyl, 2,3,6-trichlorophenyl, 2,4,5-trichlorophenyl, 3,4,5- tri chlorophenyl, 2,4,6-trichlorophenyl, 2,3-dimethylphenyl, 2,4-dimethylphenyl, 2,5- dimethylphenyl, 2,6-dimethylphenyl, 2,3,4-trimethylphenyl, 2,3,5-trimethylphenyl, 2,3,6- trimethylphenyl, 2,4,5-trimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3 -ethylphenyl, 4- ethylphenyl, 2,3 -di ethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3,4- diethylphenyl, 2, 3, 4-tri ethylphenyl, 2,3,5-triethylphenyl, 2,3,6-triethylphenyl, 2,4,5- triethylphenyl, 2,4,6-triethylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, and 4- isopropylphenyl.

[00147] Non-limiting examples of substituted aryl groups include 2-aminophenyl, 2-(N- methylamino)phenyl, 2-(7V,7V-dimethylamino)phenyl, 2-(7V-ethylamino)phenyl, 2-(N,N- diethylamino)phenyl, 3 -aminophenyl, 3-(A-methylamino)phenyl, 3-(N,N- dimethylamino)phenyl, 3-(7V-ethylamino)phenyl, 3-(A,A-diethylamino)phenyl, 4-aminophenyl, 4-(7V-methylamino)phenyl, 4-(7V,7V-dimethylamino)phenyl, 4-(7V-ethylamino)phenyl, and 4-(N,N- di ethyl amino)pheny 1.

[00148] A heterocycle can be any ring containing a ring atom that is not carbon, for example, N, O, S, P, Si, B, or any other heteroatom. A heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms. A heterocycle can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.

[00149] Non-limiting examples of heterocycles include: heterocyclic units having a single ring containing one or more heteroatoms, non-limiting examples of which include, diazirinyl, aziridinyl, azetidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolinyl, oxathiazolidinonyl, oxazolidinonyl, hydantoinyl, tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, piperidin-2-onyl, 2,3,4,5-tetrahydro-l/f-azepinyl, 2,3 -dihydro- 1/7-indole, and 1,2,3,4-tetrahydroquinoline; and ii) heterocyclic units having 2 or more rings one of which is a heterocyclic ring, non-limiting examples of which include hexahydro- 1/Z-pyrrolizinyl, 3a,4,5,6,7,7a-hexahydro-l/7- benzo[d]imidazolyl, 3a,4,5,6,7,7a-hexahydro-U7-indolyl, 1,2,3,4-tetrahydroquinolinyl, and decahydro- lZ7-cycloocta[b]pyrrolyl.

[00150] Non-limiting examples of heteroaryl include: i) heteroaryl rings containing a single ring, non-limiting examples of which include, 1,2,3,4-tetrazolyl, [l,2,3]triazolyl, [l,2,4]triazolyl, triazinyl, thiazolyl, 1/7-imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, furanyl, thiophenyl, pyrimidinyl, 2-phenylpyrimidinyl, pyridinyl, 3-methylpyridinyl, and 4-dimethylaminopyridinyl; and ii) heteroaryl rings containing 2 or more fused rings one of which is a heteroaryl ring, nonlimiting examples of which include: 77/-purinyl, 97/-purinyl, 6-amino-9J7-purinyl, 5H- pyrrolo[3,2-t ]pyrimidinyl, 7Z7-pyrrolo[2,3- ]pyrimidinyl, pyrido[2,3-t ]pyrimidinyl, 4, 5,6,7- tetrahydro- l -7/-indolyl, quinoxalinyl, quinazolinyl, quinolinyl, 8-hydroxy-quinolinyl, and isoquinolinyl.

[00151] Any compound herein can be purified. A compound herein can be at least about 1% pure, at least about 2% pure, at least about 3% pure, at least about 4% pure, at least about 5% pure, at least about 6% pure, at least about 7% pure, at least about 8% pure, at least about 9% pure, at least about 10% pure, at least about 11% pure, at least about 12% pure, at least about 13% pure, at least about 14% pure, at least about 15% pure, at least about 16% pure, at least about 17% pure, at least about 18% pure, at least about 19% pure, at least about 20% pure, at least about 21% pure, at least about 22% pure, at least about 23% pure, at least about 24% pure, at least about 25% pure, at least about 26% pure, at least about 27% pure, at least about 28% pure, at least about 29% pure, at least about 30% pure, at least about 31% pure, at least about 32% pure, at least about 33% pure, at least about 34% pure, at least about 35% pure, at least about 36% pure, at least about 37% pure, at least about 38% pure, at least about 39% pure, at least about 40% pure, at least about 41% pure, at least about 42% pure, at least about 43% pure, at least about 44% pure, at least about 45% pure, at least about 46% pure, at least about 47% pure, at least about 48% pure, at least about 49% pure, at least about 50% pure, at least about 51% pure, at least about 52% pure, at least about 53% pure, at least about 54% pure, at least about 55% pure, at least about 56% pure, at least about 57% pure, at least about 58% pure, at least about 59% pure, at least about 60% pure, at least about 61% pure, at least about 62% pure, at least about 63% pure, at least about 64% pure, at least about 65% pure, at least about 66% pure, at least about 67% pure, at least about 68% pure, at least about 69% pure, at least about 70% pure, at least about 71% pure, at least about 72% pure, at least about 73% pure, at least about 74% pure, at least about 75% pure, at least about 76% pure, at least about 77% pure, at least about 78% pure, at least about 79% pure, at least about 80% pure, at least about 81% pure, at least about 82% pure, at least about 83% pure, at least about 84% pure, at least about 85% pure, at least about 86% pure, at least about 87% pure, at least about 88% pure, at least about 89% pure, at least about 90% pure, at least about 91% pure, at least about 92% pure, at least about 93% pure, at least about 94% pure, at least about 95% pure, at least about 96% pure, at least about 97% pure, at least about 98% pure, at least about 99% pure, at least about 99.1% pure, at least about 99.2% pure, at least about 99.3% pure, at least about 99.4% pure, at least about 99.5% pure, at least about 99.6% pure, at least about 99.7% pure, at least about 99.8% pure, or at least about 99.9% pure.

Pharmaceutically-acceptable salts.

[00152] The disclosure provides the use of pharmaceutically-acceptable salts of any therapeutic compound described herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically-acceptable salt is an ammonium salt.

[00153] Metal salts can arise from the addition of an inorganic base to a compound of the disclosure. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[00154] In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

[00155] Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the disclosure. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N- methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.

[00156] In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or a pipyrazine salt.

[00157] Acid addition salts can arise from the addition of an acid to a compound of the disclosure. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

[00158] In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccarate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt.

Fluorosequencing

[00159] Fluorosequencing or the use of fluorescent probes for amino acid identification can refer to sequencing polypeptides in one or more analytes (e.g, protein sample comprising a polypeptide or a peptide) at the level of single molecules. In some cases, a plurality (e.g., millions) of individual fluorescently labeled peptides can be visualized in parallel, monitoring changing patterns of one or more fluorescent properties (e.g., fluorescence intensity, emission spectra, or fluorescent lifetime) as the terminal amino acids (e.g., C-terminal amino acid or N- terminal amino acids) are removed, and/or using the resulting fluorescence signatures (fluorosequences) to uniquely identify individual polypeptide. In some cases, one or more amino acids may be labeled in a selective manner on polypeptides or peptides. In some cases, one or more amino acids may be labeled globally (e.g., a universal probe) on polypeptides or peptides based on its position (e.g., N-terminus or C-terminus). The polypeptides or peptides can be immobilized to a support. The amino acids can undergo iterative cycles of removing terminal residues and/or analyzing (e.g, imaging) corresponding changes in fluorescent intensity for individual peptide molecules. Methods, systems, compositions, and/or kits disclosed herein can generate profiles or patterns adequately representative of the polypeptide sequences to results in unique identification of one or more (e.g., plurality) of proteins from a particular genus or specie The resulting changes in one or more fluorescent characteristics (e.g., fluorescent emission or lifetime) may provide identification of the amino acid residues (e.g., amino acid residues at C- or N- terminus or an internal amino acid residues). In some cases, the single molecule technologies of the present disclosure allow the identification and/or absolute quantitation of a given peptide or protein in a biological sample. In some cases, the methods described herein can be used to identify one or more modifications to the polypeptides or at least a portion of the polypeptide.

[00160] In some cases, the methods, systems, compositions, and/or kits disclosed herein can be used to perform large- scale sequencing of a polypeptide, peptide, or a plurality of single intact peptides (denatured or not denatured) at the single molecule level by selective labeling amino acids in fluid or on immobilized peptides followed by successive cycles of labeling and/or removal of the peptide amino terminal amino acids. In some cases, the methods, systems, compositions, and/or kits disclosed herein can be used to perform large- scale sequencing of a polypeptide, peptide, or a plurality of single intact peptides (denatured or not denatured) at the single molecule level by selective labeling terminal amino acids in fluid or on immobilized peptides followed by successive cycles of labeling and/or removal of the peptide amino terminal amino acids. The methods, systems, compositions, and/or kits disclosed herein can identify the amino acids in polypeptides, including polypeptides comprising natural amino acids and/or unnatural amino acids. In some cases, methods, systems, compositions, and/or kits disclosed herein can comprise labeling the amino acid (e.g., N-terminal, C-terminal, or internal amino acid) with a probe. In other cases, the methods, systems, compositions, and/or kits disclosed herein can comprise labeling the amino acid (e.g., N-terminal, C-terminal, or internal amino acid) with one or more probes. In some cases, a probe of one or more probes can specifically bind to at least one amino acid of the polypeptide.

[00161] Various aspects of the present disclosure provide methods, systems, compositions, and/or kits disclosed herein for polypeptide fluorosequencing, also called sequencing by degradation. For example, a molecule (e.g., a polypeptide) may be labeled with a probe, then subsequently digested and/or subjected to fluorosequencing for sequencing analysis. In some cases, present disclosure may provide a massively parallel and/or rapid method for identifying and/or quantitating individual polypeptide and/or protein molecules within a given complex sample.

[00162] In another aspect, the present disclosure provides methods for determining at least one characteristic of at least a portion of one or more analytes. In another aspect, the present disclosure provides methods for determining at least one characteristic (e.g., sequence) of at least a portion of the polypeptide. The analyte may comprise a protein, polypeptide, or peptide. The present disclosure may also provide methods for removing a terminus of the analyte. The terminus may be an N-terminus or C-terminus of the analyte.

[00163] The method can comprise providing one or more analytes comprising one or more polypeptides. In some cases, the polypeptide (e.g., of one or more polypeptides) may comprise one or more probes coupled to one or more amino acids of the polypeptide. For example, in some cases, one or more probes may be coupled to an N-terminal amino acid (of the polypeptide). In some cases, one or more probes may be coupled to a C-terminal amino acid. In other cases, one or more probes may be coupled to one or more internal amino acids. In some cases, the one or more probes described herein may be configured to generate one or more detectable signals or signal change (e.g., fluorescent spectral properties) when coupled to one or more amino acids of the polypeptide. In some cases, the method can comprise detecting one or more signals or signal change from the one or more probes. For example, in some cases, the one or more probes coupled to one or more amino acids of the polypeptide can generate one or more signals or signal change that are unique to amino acids. In some cases, the one or more signals or signal change that are unique to the amino acids can be used to identify at least one characteristic of the at least a portion of the polypeptide. In some cases, the detection of one or more signals or signal change may occur before degrading one or more amino acids (e.g., one or more amino acids coupled to one or more probes) from the polypeptide. In some cases, the detection of one or more signals or signal change occurs after degrading one or more amino acids (e.g., one or more amino acids coupled to one or more probes) from the polypeptide. For example, in some cases, the detection may use at least one fragment comprising the amino acid residue (e.g., coupled to the probe) that has been cleaved from the polypeptide to identify at least one characteristic of the at least the portion of the polypeptide. In some cases, the one or more probes, after detecting, may be degraded (e.g., cleaved) from the one or more amino acids of the polypeptides. In some cases, the one or more probes may be subjected to one or more conditions sufficient to degrade the one or more probes from one or more amino acids of the polypeptide. For example, in some cases, the one or more conditions may comprise, but not limited to, Edman, or related, chemical degradation, enzymatic degradation, an optical condition, or any other suitable methods. In some cases, the one or more probes, after detecting, may not be degraded, but still bound to the one or more amino acids (e.g., coupled to the one or more amino acids of the polypeptides). In some cases, after detecting, a terminal amino acid of the polypeptide may be cleaved from the polypeptide. In some cases, upon detecting, the methods described herein may comprise subjecting at least a portion of the polypeptide to one or more conditions to remove a terminal amino acid from the polypeptide. In some cases, the one or more conditions may be sufficient to generate a modified polypeptide. In some cases, the modified polypeptide may comprise one or more amino acids fewer than the polypeptide. In some cases, the one or more conditions sufficient to generate a modified polypeptide may include a buffer condition, a pH condition, an optical condition, a duration condition, temperature, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may be an optical condition (e.g., light). In some cases, the optical condition may comprise subjecting at least a portion of the polypeptide to one or more light sources. In some cases, the one or more conditions may be applied to at least a portion of the polypeptide sequentially or at the same time. In other cases, the at least a portion of the polypeptide may be subjected to at least two conditions sufficient to generate a modified polypeptide. In some cases, the at least two conditions may be the same conditions with different properties (e.g., different pH, different temperatures, light with different wavelengths, different buffers, and/or different durations). For example, the methods described herein may comprise subjecting at least a portion of the polypeptide to (1) light with first wavelength, and, subsequently or at the same time, (2) an additional light with second wavelengths. In some cases, light with the first wavelengths may be sufficient to remove the one or more probes (e.g., one or more probes coupled to one or more amino acids of the polypeptide). In some cases, the additional light with second wavelengths may be sufficient to remove one or more amino acids (e.g., a terminal amino acid) of the polypeptide. In some cases, the at least a portion of the polypeptide may be subjected to at least two different conditions sufficient to generate a modified polypeptide. For example, the methods described herein may comprise subjecting at least a portion of the polypeptide to (1) a first condition comprising a pH condition (e.g., a pH that is at least about 9) and (2) a second condition comprising light with one or more wavelengths.

[00164] In some cases, the one or more conditions may be exposing at least a portion of the polypeptide to light. In some cases, the light can be directed onto at least a portion of an analyte (e.g., polypeptide). In some cases, the light can be directed to a plurality of analytes. In some cases, the light can be directed to at least a portion of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, less than all of the analytes may come into contact with one or more light sources. In some cases, the light may be directed to the entirety of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, all of the analytes may come into contact with one or more light sources. In some cases, the light can be directed to at least a portion of a polypeptide that is coupled to a support. In some cases, the light can be directed to an entire solution comprising one or more analytes. In some cases, the light can be directed to at least a portion of a solution comprising one or more analytes. If a plurality of analytes in solution, less than all of the analytes may come into contact with one or more light sources.

[00165] In some cases, a terminus (e.g., N-terminus or C-terminus) of one or more analytes comprising a polypeptide may be contacted with a degradation agent disclosed herein. In some cases, the degradation agent disclosed herein may comprise a photo-cleavable moiety. Upon contacting the polypeptide with a degradation agent, a first modified polypeptide may be formed. In some cases, upon contacting the polypeptide with a degradation, a first modified polypeptide comprising a photo-cleavable moiety may be formed. In some cases, the photo- cleavable moiety may be cleavable when subjected to one or more one or more conditions, thereby forming a second modified polypeptide. In other cases, the second modified polypeptide may have one or more fewer amino acids that the polypeptide. For example, one or more conditions sufficient to generate a second modified peptide may comprise subjecting the first modified polypeptide with a buffer condition, a pH condition, an optical condition, a duration condition, temperature, water solubility of the degradation agent, or any combination thereof. In some cases, the photo-cleavable moiety of the first modified polypeptide may be cleavable when subjected to light (e.g., an optical condition). In some cases, the first modified polypeptide may be subjected to at least two conditions sufficient to generate the second modified polypeptide. For example, in some cases, the first condition may be subjecting to light, while the second condition may be any other conditions selected from the group consisting of a pH condition, an optical condition, a duration condition, temperature, water solubility of the degradation agent, and any combination thereof. In some cases, at least a portion of the first modified polypeptide may be subjected to one or more conditions sufficient to generate a fragment comprising a residue of the terminus of the polypeptide. The first modified polypeptide (e.g., at least a portion of the first modified polypeptide) may be subjected to one or more conditions sufficient to generate a second modified polypeptide and/or a fragment comprising a residue of the terminus of the polypeptide. In some cases, the first modified polypeptide (e.g., at least a portion of the first modified polypeptide) may be in a solution when one or more conditions are applied. In some cases, the solution may comprise one or more acids (e.g., Lewis acid), one or more bases, one or more chelators, one or more catalysts, one or more enzymes, one or more solvents (e.g., DMSO), one or more drying agents, one or more salts, and/or one or more scavengers. In some cases, the first modified polypeptide (e.g., at least a portion of the first modified polypeptide) may be subjected to a solution comprising one or more acids (e.g., Lewis acid) to generate a second modified polypeptide. In some cases, the first modified polypeptide (e.g., at least a portion of the first modified polypeptide) may be subjected to light comprising one or more wavelengths to generate a second modified polypeptide.

[00166] The second modified polypeptide can comprise one or more fewer amino acids than the polypeptide. In some cases, the second modified polypeptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20, or more amino acids fewer than the polypeptide. In some cases, the second modified polypeptide may comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 amino acid(s) fewer than the polypeptide.

[00167] In some cases, the contacting one or more probes to the polypeptide, the detecting one or more signals or signal change, and/or the contacting a terminus of a polypeptide with a degradation agent to degrade one more amino acids (e.g., a terminal amino acid), may be repeated. For example, in some cases, the methods described herein may be repeated at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more cycles.

[00168] In some cases, the methods, compositions, systems, and/or kits described herein may be used to determine a plurality of characteristics of a plurality of polypeptides in parallel or at least a portion of the polypeptide of the plurality of polypeptide in parallel. In some cases, at least one polypeptide may be provided. The at least one polypeptide may comprise a plurality of polypeptides. In some cases, one or more characteristics may be determined for each of the plurality of polypeptides according to any method disclosed herein. The plurality of polypeptides may be the entirety of polypeptides in a sample. In other cases, the plurality of polypeptides may be less than all of the polypeptides in the sample. The sample may comprise the plurality of polypeptides and one or more additional polypeptides (e.g., a first polypeptide or a second polypeptide). For example, less than all of the polypeptides in a sample may be analyzed according to any method disclosed herein, and/or using any system, composition, or kit disclosed herein. As another example, each of the polypeptides in the sample may be analyzed according to any method disclosed herein, and/or using any system, composition, or kit disclosed herein.

[00169] In some cases, the plurality of polypeptides may be characterized, while the one or more additional polypeptides may not be characterized. The one or more additional polypeptides may not be contacted with one or more probes disclosed herein, and/or one or more degradation conditions disclosed herein (e.g., degradation agent, light conditions). [00170] In another aspect, provided herein is a sample comprising at least a plurality of polypeptides. A polypeptide of the plurality of polypeptides may be coupled to one or more probes. For example, in some cases, a first polypeptide is coupled to first one or more probes. In some cases, a second polypeptide may be coupled to second one or more probes. In some cases, the first one or more probes and the second one or more probes are the same (e.g., have the same chemical structure). For example, in some cases, the first one or more probes and the second one or more probes may be coupled to N-terminal amino acids. In other cases, one or more probes or the second one or more probes may be different. For example, the first one or more probes may be coupled to N-terminal amino acids, while the second one or more probes may be coupled to specific amino acids (e.g., lysine-specific probe) or may be coupled to amino acid types (e.g., non-natural amino acids, hydrophobic acids, aromatic amino acids, or amino acids based on positional classification). In another example, the first one or more probes may be coupled to specific amino acids (e.g., lysine-specific probe), while the second one or more probes may be coupled to another specific amino acids (e.g., cysteine-specific probe). In some cases, the one or more probes, when coupled to one or more amino acids, may be configured to generate one or more signals or signal change. In some cases, the methods described herein may comprise detecting one or more signals or signal change from the one or more probes coupled to the polypeptide (e.g., one or more probes coupled to one or more amino acids of the polypeptide) of the plurality of polypeptides. In some cases, a terminus (e.g., N-terminus or C-terminus) of the plurality of polypeptides may be coupled to one or more degradation agents. For example, in some cases, a terminus of the first polypeptide may be contacted with a first degradation agent. In some cases, a terminus of the second polypeptide may be contacted with a second degradation agent. In some cases, the first degradation agent and the second degradation agent may be the same degradation agent (e.g., a degradation agent described herein). For example, in some cases, the first degradation agent and the second degradation agent can be the degradation agent comprising photo-cleavable moiety. In other cases, the first degradation agent and the second degradation agent may be different. For example, in some cases, the first degradation agent may be a degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety), and the second degradation agent may be an Edman degradation agent (e.g., phenylisothiocyanate), an enzymatic and/or chemical cleavage agent (e.g., cyanogen bromide, pepsin, or thermolysin), anhydrous hydrazine, dansyl chloride, or any combination thereof. In some cases, the methods described herein may comprise identifying one or more characteristics of the polypeptide or a plurality of polypeptides. In some cases, the methods described herein may comprise identifying one or more characteristics of an analyte or a plurality of analytes. In some cases, the methods described herein may comprise identifying one or more characteristics of a sample or a plurality of samples. In some cases, the one or more characteristics of the polypeptide may include identification of the one or more amino acids of the polypeptide, modifications of the one or more amino acids of the polypeptide. In some cases, the one or more characteristics of the sample may include, but are not limited to, a number of polypeptides in the sample, type of polypeptide in the sample, an origin of the sample, impurities in the sample, presence of a polypeptide, absences of a polypeptide, or any combinations thereof. In other cases, the one or more characteristics of the polypeptide may be the quantity of the polypeptide present in a sample. In some cases, the one or more characteristics of the sample may be obtained by sequencing a plurality of polypeptides from one or more samples.

[00171] In some cases, an analyte disclosed herein may be among a sample. One or more characteristics of the sample can be determined using one or more properties of at least a portion of the analyte and/or at least another analyte. The one or more characteristics of the sample may comprise an absolute or relative abundance, absolute concentration, an absolute or relative abundance, origin of one or more analyte types in the sample, or any combination thereof. The one or more characteristics can comprise a relative concentration, absolute concentration, absolute or relative abundance, origin of one or more analytes in the sample, or any combination thereof. The one or more properties may comprise a quantity or percentage of different and/or modified analytes in the plurality of analytes. The one or more characteristics may comprise determining differences in identity or sequence (e.g., differences in sequence is of at most 15 amino acids, at most 10 amino acids, etc.) of at most 20 units between at least a subset of analytes of the plurality of analytes. In other cases, analyzing the one or more characteristics comprises determining the quantity of analytes in a first type of analyte and determining a quantity of analytes in at least a second type of analyte. In the first type of analyte and/or at least the second type of analyte, one or more of relative abundance of analytes in the sample, analytes with secondary structures, one or more impurities in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with tertiary structures, a number of analytes, analytes with quaternary structures, or any combination thereof may be determined.

[00172] In another aspect, the methods described herein may comprise detecting one or more signals or signal change. In some cases, the one or more signals or signal change may be generated by one or more probes coupled to a polypeptide or an amino acid. In some cases, at least a portion of the polypeptide may be subjected to one or more conditions. The one or more conditions may be sufficient to generate a modified polypeptide. In some cases, the modified polypeptide may comprise one or more amino acids (e.g., a terminal amino acid) fewer than the polypeptide. In some cases, at least a portion of the polypeptide may be subjected to a light condition. In some cases, the light condition may be sufficient to remove a terminal amino acid of the polypeptide. In some cases, the one or more signals or signal change may be used to determine one or more characteristics of the at least the portion of the analyte (e.g, comprising one or more polypeptides). In some cases, the one or more signals or signal change may be used to determine one or more characteristics of the at least the portion of the analyte (e.g., polypeptide) with an accuracy of about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 92 %, about 94 %, about 96 %, about 98 %, or about 99 %. In some cases, the one or more signals or signal change may be used to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 92 %, at least about 94 %, at least about 96 %, at least about 98 %, at least about 99%, or more. In some cases, the one or more signals or signal change may be used to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at most about 65 %, at most about 70 %, at most about 75 %, at most about 80 %, at most about 85 %, at most about 90 %, at most about 92 %, at most about 94 %, at most about 96 %, at most about 98 %, at most about 99 % or less. One or more characteristics of an analyte disclosed herein can be determined with a degree of accuracy. In some cases, the analyte characteristic can comprise a sequence associated with the analyte. The accuracy can be evaluated as a consensus accuracy, a sequence accuracy, an identity, as an amino acid accuracy, or any combinations thereof.

[00173] In some cases, a plurality of characteristics can be analyzed to improve identification of an analyte or the characteristic. In some embodiments, signal or signal change thereof can be combined across many analytes to improve determining the analyte’s characteristic.

[00174] In some cases, the methods, systems, kits, compositions, or any combination thereof described herein may be utilized to determine a characteristic associated with the analyte with a high accuracy. The one or more signals or signal changes can be used to determine the one or more characteristics of any analyte (e.g., polypeptide) disclosed herein with an accuracy. The accuracy of determining the one or more characteristics of the analyte may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. The accuracy of identifying the one or more characteristics of the analyte may be at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20%.

[00175] In some cases, a sequence accuracy can be a percentage accuracy of an analyte sequence obtained from the methods, compositions, kits, or systems disclosed herein as compared to a reference sequence associated with the analyte. In other cases, a consensus accuracy of any analyte disclosed herein can be determined by comparing one or more reads of an analyte to one or more references. In some examples, a consensus accuracy can be determined by at least at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or greater than about 10 reads from an analyte. In some cases, a consensus accuracy may be obtained by comparing a plurality of analytes to one or more references and/or obtaining at least blended accuracy, blended score, cumulative median, blended probability, cumulative probability, a blended, cumulative average, median, cumulative accuracy, or cumulative score across the multiple analytes. In some cases, a consensus accuracy can be obtained from a single molecule if it is read multiple times in a multi-pass manner across the nanopore.

[00176] In some cases, a sequence can be determined with an amino acid accuracy of about 50% to about 99.9%. In some cases, the sequence can be determined with an amino acid accuracy of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.9%. In some cases, a sequence can be determined with an amino acid accuracy of 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%. [00177] In other cases, a sequence can be determined with a consensus accuracy of about 50% to about 99.9%. In some cases, the sequence can be determined with a consensus accuracy of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.9%. In some cases, a sequence can be determined with a consensus accuracy of about 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%.

[00178] In some cases, a sequence can be determined with a sequence accuracy of about 50% to about 99.9%. In some cases, the sequence can be determined with a sequence accuracy of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.9%. In some cases, a sequence can be determined with a sequence accuracy of 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%.

[00179] In some cases, an analyte can be determined with a specificity. In some cases, an analyte can be determined with a specificity of at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.9%. In some embodiments, an analyte can be determined with a true negative rate of at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 98%, at most about 99%, at most about 99.9%, or at most about 100%.

[00180] In another aspect, provided herein are methods for analyzing one or more samples or determining one or more characteristics of an analyte (e.g., a polypeptide). In some cases, the polypeptide and at least one additional polypeptide may be provided. In some cases, the polypeptide and the at least one additional polypeptide may be from the same sample. In some cases, the polypeptide and the at least one additional polypeptide may be from different samples. In some cases, the polypeptide may be provided at a first location on a first support. The at least one additional polypeptide may be provided at an additional location on an additional support. The polypeptide may be coupled to one or more probes. In other cases, the at least one additional polypeptide may be coupled to one or more probes. In some cases, the one or more probes, when coupled to the polypeptides (e.g., when coupled to one or more amino acids of the polypeptides) and/or to the at least one additional polypeptide, may be configured to generate one or more signals or signal change. In some cases, the one or more signals or signal changes may be unique to the one or more amino acids that are bound to one or more probes of the polypeptides or of the at least one additional polypeptide. In some cases, the methods described herein may comprise detecting one or more signals or signal change from the one or more probes of the polypeptide.

[00181] In some cases, the methods described herein may further comprise subjecting the polypeptide and/or the additional polypeptide (e.g., that is coupled to one or more probes or one or more additional probes, respectively) to one or more conditions sufficient to generate a modified polypeptide and/or a modified additional polypeptide. In some cases, the modified polypeptide and/or the modified additional polypeptide may comprise one or more amino acids fewer than the polypeptide or the additional polypeptide, respectively. In some cases, the one or more conditions sufficient to generate the modified polypeptide and/or the modified additional polypeptide may comprise a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may be selectively applied to the polypeptide, but not to the additional polypeptide. In some cases, the one or more conditions may be selectively applied to the additional polypeptide, but not to the polypeptide. In some cases, one or more conditions may be applied to the polypeptide or the additional polypeptide may be different. For example, the polypeptide may be subjected to a first condition, while the additional polypeptide may be subjected to a second condition that is different from the first condition. In some cases, the polypeptide may be subjected to light having a first wavelength, while the additional polypeptide may be subjected to light having a second wavelength. In some cases, the light having the first wavelength and the light having the second wavelength may differentially affect the polypeptide and/or the additional polypeptide. For example, in some cases, the light having the first wavelength may cause one or more probes to be cleaved from the polypeptide and/or the additional polypeptide, thereby exposing the one or more amino acids (that were previously bound to the one or more probes). In some cases, the light having the second wavelength may cause one or more amino acids to be cleaved from the polypeptide and/or the additional polypeptide, thereby exposing the next amino acids (e.g., exposing the next N-terminal amino acids or C-terminal amino acids) of the polypeptide and/or the additional polypeptide.

[00182] In some cases, the one or more probes coupled to the polypeptide may be subjected to light comprising one or more wavelengths. In some cases, the one or more probes coupled to the additional polypeptide may not be subjected to light comprising one or more wavelengths. In some cases, methods provided herein may selectively expose at least a portion of the plurality of polypeptides (e.g., comprising the polypeptide and at least one additional polypeptide) to light while shielding other areas from such exposure. This selective illumination may be achieved using photomasks, a waveguide, a spatial light modulator, or digital micromirror devices. For example, in some cases, the methods may selectively subject at least a portion of the first polypeptide to light comprising one or more wavelengths. In some cases, the selective illumination may be achieved using one or more light sources. In some cases, the one or more light source may be used with one or more photomasks, waveguides, spatial light modulators, or digital micromirror devices. In some cases, the one or more light sources may be configured to emit electromagnetic radiation in the ultraviolet, visible, or infrared spectrum. In some cases, the one or more light sources may be employed to initiate, modulate, or facilitate a photoreaction, photodegradation, fluorescence excitation, or other optically responsive processes described herein. In some cases, the light source may include, without limitation: light-emitting diodes (LEDs), laser diodes, solid-state lasers, gas lasers, halogen lamps, incandescent bulbs, and/or sunlight.

[00183] In some cases, the methods may comprise selectively providing the light to the at least the portion of the polypeptide at the first location of the first support. In some cases, the methods may comprise selectively providing the light to the at least the portion of the polypeptide at the first location of the first support, but not providing the light to the at least the portion of the at least one additional polypeptide at the additional location of the additional support. In some cases, the methods may comprise selectively providing the light to the at least the portion of the at least one additional polypeptide at the additional location of the additional support. In some cases, the methods may comprise selectively providing the light to the at least the portion of the at least one additional polypeptide at the additional location of the additional support, but not providing the light to the at least the portion of the polypeptide at the location of the support. In some cases, the methods may comprise selectively subjecting the at least the portion of the polypeptide to the light at a first time and subjecting at least a portion of the second polypeptide to another light at a second time. In some cases, a distance between the first location and the additional location can be at least about 50 nm, at least about 80 nm, at least about 100 nm, at least about 120 nm, at least about 150 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 1 mm, at least about 5 mm, at least about 10 mm, at least about 20 mm, at least about 30 mm, at least about 40 mm, at least about 50 mm, at least about 100 mm, at least about 500 mm, at least about 1 cm, at least about 5 cm, at least about 10 cm or more. In some cases, a distance between the first location and the additional location can be at most about 50 nm, at most about 80 nm, at most about 100 nm, at most about 120 nm, at most about 150 nm, at most about 200 nm, at most about 300 nm, at most about 400 nm, at most about 500 nm, at most about 1 mm, at most about 5 mm, at most about 10 mm, at most about 20 mm, at most about 30 mm, at most about 40 mm, at most about 50 mm, at most about 100 mm, at most about 500 mm, at most about 1 cm, at most about 5 cm, at most about 10 cm or more.

[00184] In some cases, the light (e.g., the light comprising one or more wavelengths) may selectively remove a terminal amino acid of the polypeptide, while leaving a terminal amino acid of the at least one additional polypeptide intact. The at least one additional polypeptide may not be characterized. The at least one additional polypeptide may not be contacted with one or more probes disclosed herein, and/or one or more degradation conditions disclosed herein (e.g., degradation agent, light conditions). In some cases, the methods may be repeated such that only the polypeptide may successfully undergo (1) detection step or operation (e.g., detecting one or more signals) and (2) removal step or operation (e.g., removing one or more amino acids from the polypeptide), while the additional polypeptide is intact.

[00185] For example, in some cases, the methods, compositions, systems, and/or kits described herein may comprise providing a first polypeptide and a second polypeptide, wherein the first polypeptide is coupled to a first probe, and the second polypeptide is coupled to a second probe. In some cases, the first probe and the second probe may be coupled to a terminal amino acid of the first polypeptide and the second polypeptide. In some cases, upon conjugation, the methods further comprise detecting one or more signals or signal change from the first polypeptide and/or the second polypeptide. In some cases, the methods may comprise selectively subjecting at least a portion of the first polypeptide to one or more conditions sufficient to generate a first modified polypeptide. In some cases, the one or more conditions may be sufficient to remove a terminal amino acid of the first polypeptide, exposing a next terminal amino acid of the first polypeptide. In some cases, the second polypeptide may not be subjected to the one or more conditions. In some cases, the first modified polypeptide may be subjected to a second cycle of the methods described herein. For example, in some cases, the first modified polypeptide may be coupled to a probe (e.g., a new probe having the same chemical structure as the first probe). In some cases, one or more signals or signal change may be detected. Upon detection, the first modified polypeptide may be subjected to one or more conditions sufficient to remove the newly exposed terminal amino acid. In some cases, the second polypeptide may not be subjected to the one or more conditions and may still be intact. Subsequently and/or independently, the second polypeptide (e.g., the second polypeptide) may be exposed to one or more conditions sufficient to remove a terminal amino acid, thereby forming a second modified polypeptide exposing a next terminal amino acid. In some cases, the second modified polypeptide may be coupled to a probe (e.g., a new probe having the same chemical structure as the second probe). In some cases, one or more signals or signal change may be detected from the second modified polypeptide. Upon detection, the second modified polypeptide may be subjected to one or more conditions sufficient to remove the newly exposed terminal amino acid. In some cases, the first modified polypeptide may not be subjected to the one or more conditions and may still be intact. [00186] In another example, in some cases, the methods, compositions, systems, and/or kits described herein may comprise providing a first polypeptide and a second polypeptide, wherein the first polypeptide is coupled to a first probe, and the second polypeptide is coupled to a second probe. In some cases, the first probe and the second probe may be coupled to a terminal amino acid of the first polypeptide and the second polypeptide. In some cases, upon conjugation, the methods further comprise detecting one or more signals or signal change from the first polypeptide and/or the second polypeptide. In some cases, the first polypeptide and/or the second polypeptide may be exposed to one or more conditions sufficient to remove the first probe and the second probe (from the one or more amino acids that were previously coupled to the first probe and/or the second probe). Upon removal of the first probe and the second probe, the first probe and/or the second probe may be further coupled to a degradation agent described herein. For example, in some cases, the degradation agent described herein may comprise a photo-cleavable moiety. In some cases, the degradation agent that is coupled to the first polypeptide and/or the second polypeptide can only degrade a terminal amino acid only upon exposure to one or more conditions. In some cases, the methods may comprise selectively subjecting at least a portion of the first polypeptide to one or more conditions sufficient to remove a terminal amino acid from the first polypeptide, thereby generating a first modified polypeptide. In some cases, the one or more conditions may be sufficient to remove a terminal amino acid of the first polypeptide, exposing a next terminal amino acid of the first polypeptide. In some cases, the second polypeptide may not be subjected to the one or more conditions. In some cases, the first modified polypeptide may be subjected to a second cycle of the methods described herein. For example, in some cases, the first modified polypeptide may be coupled to a probe (e.g., a new probe having the same chemical structure as the first probe). In some cases, one or more signals or signal change may be detected. Upon detection, the first modified polypeptide may be exposed to one or more conditions sufficient to remove the probe from the first modified polypeptide. In some cases, upon removal of the probe, the first modified polypeptide may be subjected to one or more conditions sufficient to remove the newly exposed terminal amino acid. In some cases, the second polypeptide may not be subjected to the one or more conditions and may still be intact. Subsequently and/or independently, the second polypeptide (e.g., the second polypeptide) may be coupled to a degradation agent described herein. When exposed to one or more conditions sufficient to remove a terminal amino acid, a second modified polypeptide exposing a next terminal amino acid may be formed. In some cases, the second modified polypeptide may be coupled to a probe (e.g., a new probe having the same chemical structure as the second probe). In some cases, one or more signals or signal change may be detected from the second modified polypeptide. Upon detection, the second modified polypeptide may be subjected to one or more conditions sufficient to remove the newly exposed terminal amino acid. In some cases, the first modified polypeptide may not be subjected to the one or more conditions and may be intact.

[00187] In another aspect, provided herein are methods to detect at least one characteristic of one or more samples or one or more polypeptides. In some cases, the methods may comprise providing one or more polypeptides (e.g., a polypeptide and/or an additional polypeptide). In some cases, the one or more polypeptides may be coupled to one or more probes (e.g., a probe and/or an additional probe). The probe and/or the additional probe may be coupled to N-terminal amino acids or C-terminal amino acids. In some cases, the one or more probes, when bound to one or more amino acids of the polypeptide and/or the additional polypeptide may be configured to produce one or more signals or signal changes that are unique to the one or more amino acids bound to the one or more probes. In some cases, the methods described herein may comprise detecting one or more signals or signal change from the one or more probes coupled to the polypeptide and/or the additional polypeptide. Upon detection, the one or more probes that are bound to the polypeptide and/or the additional polypeptide (e.g., bound to one or more amino acids of the polypeptide and/or one or more amino acids of the additional polypeptide) may be cleaved using light, chemical, enzymatic, photolytic, thermal treatment, or any combination thereof. The cleavage of the one or more probes may be performed under conditions that do not alter the chemical integrity of the underlying amino acid residues, thereby leaving the amino acids (e.g., of the terminal amino acids of the polypeptide or the additional polypeptide) substantially unmodified. For example, the one or more amino acids of the polypeptide and/or the additional polypeptide that are previously bound to the one or more probes may be regenerated in their native or functional form upon exposure to one or more conditions sufficient to remove the one or more probes. The one or more conditions sufficient to remove oen or more probes may be light, chemical, enzymatic, photolytic, thermal treatment, or any combination thereof. Upon removal of the one or more probes, the terminal amino acid of the polypeptide and/or the terminal amino acid of the additional polypeptide may be removed when subjected to one or more conditions (e.g., light, degradation agent). For example, upon removal of the one or more probes, the polypeptide and/or the additional polypeptides may be subjected to one or more conditions sufficient to remove the terminal amino acid. In some cases, the one or more conditions sufficient to remove the one or more amino acid from the polypeptide or the additional polypeptide may be a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, a degradation agent, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may comprise a degradation agent described herein. In some cases, the one or more conditions may include conjugating the polypeptide and/or the additional polypeptide to a degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety). For example, in some cases, the one or more degradation agents may comprise a photo-cleavable moiety. In some cases, upon coupling the one or more degradation agents to the polypeptide and/or the additional polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N-terminal amino acid) may be cleaved, exposing the next terminal amino acid (e.g., n-1 amino acid), thereby forming a modified polypeptide. In some cases, one or more methods described herein may be repeated. For example, in some cases, the modified polypeptide (e.g., having an amino acid fewer than the original polypeptide having the next amino acid, n-1 amino acid, at the terminus) may then be coupled to one or more probes (e.g., a probe and/or an additional probe) in a second cycle. The probe and/or the additional probe may be coupled to N-terminal amino acids or C-terminal amino acids of the modified polypeptide. In some cases, the one or more probes, when bound to one or more amino acids of the modified polypeptide and/or the modified additional polypeptide may be configured to produce one or more signals or signal changes that are unique to the one or more amino acids bound to the one or more probes. In some cases, the methods described herein may comprise detecting one or more signals or signal change of the one or more probes coupled to the modified polypeptide and/or the additional modified polypeptide. Upon detection, the one or more probes that are bound to the modified polypeptide and/or the additional modified polypeptide may be cleaved using light, chemical, enzymatic, photolytic, thermal treatment, or any combination thereof. Upon removal of the probes, the terminal amino acid of the polypeptide and/or the terminal amino acid of the additional modified polypeptide may be removed when subjected to one or more conditions (e.g., light, degradation agent) sufficient to remove the terminal amino acid. For example, upon removal of the one or more probes, the modified polypeptide and/or the modified additional polypeptides may be conjugated to one or more degradation agents. For example, in some cases, the one or more degradation agents may comprise a photo-cleavable moiety. In some cases, upon subjecting the one or more degradation agents coupled to the modified polypeptide and/or the additional modified polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N-terminal amino acid) may be cleaved, exposing the next amino acid (e.g., n-2 amino acid) to the terminus (e.g., N-terminus). In some cases, (1) the conjugation of one or more probes to the polypeptide or the additional polypeptide, (2) removing step or operation (e.g., removal of the one or more probes bound to the polypeptide and/or the additional polypeptide), (3) detection step or operation, (4) conjugation of the degradation agent, and/or (5) removal of the terminal amino acid of the polypeptide can be successively repeated from about 1 time to about 5 times, from about 5 times to about 10 times, from about 10 times to about 20 times, from about 20 times to about 30 times, from about 30 times to about 40 times, from about 40 times to about 50 times, from about 50 times to about 60 times, from about 60 times to about 70 times, from about 70 times to about 80 times, from about 80 times to about 90 times, or from about 90 times to about 100 times. In some cases, the methods described herein may be repeated at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, or more. In some cases, the methods described herein may be repeated at most about 5 times, at most about 10 times, at most about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times, or less. These operations and processes can be used to determine (1) at least one characteristic (or property) of the polypeptide and/or additional polypeptide and/or (2) at least one characteristic of a sample comprising the polypeptide and/or additional polypeptide.

[00188] In another aspect, provided herein are methods of detection at least one characteristic (e.g., sequence) of one or more samples or one or more polypeptides. In some cases, the methods may comprise providing one or more polypeptides (e.g., a polypeptide and an additional polypeptide). In some cases, the one or more polypeptides may be coupled to one or more probes (e.g., a probe or an additional probe). In some cases, the probe or the additional probe may be coupled to specific amino acids. For example, the probe may be coupled to any lysine residues (e.g., N-terminal lysine residues, C-terminal lysine residues, and/or internal lysine residues) of the polypeptide and/or the additional polypeptide, while the additional probe may be coupled to any cysteine residues (e.g., N-terminal cysteine residues, C-terminal cysteine residues, and/or internal cysteine residues) of the polypeptide and/or the additional polypeptide. In some cases, the one or more probes, when coupled to one or more amino acids of the polypeptide or the additional polypeptide may be configured to produce one or more signals or signal changes. In some cases, the methods described herein may comprise detecting one or more signals or signal change (or no detectable signal or signal change when the probe is not associated with the terminal amino acid of the polypeptide and/or the additional polypeptide). In some cases, the one or more signals or signal change may be indicative of the presence and/or identity of the amino acid residue. For example, in some cases, a first probe (specifically coupled to lysine) or a second probe (specifically coupled to cysteine) may be coupled to the N- terminal amino acid (e.g., lysine residue) of the polypeptide, while the N-terminal amino acid (amino acid other than lysine or cysteine) of the additional polypeptide may not be coupled to the first probe nor the second probe. Upon detection, the terminus (e.g., N-terminus or C- terminus) of the polypeptide and/or the additional polypeptide may be cleaved when exposed to one or more conditions. In some cases, the one or more conditions sufficient to remove the one or more amino acid from the polypeptide may be a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, a degradation agent, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may include conjugating the polypeptide and/or the additional polypeptide to a degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety). In some cases, upon subjecting the one or more degradation agents coupled to the polypeptide and/or the additional polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N-terminal amino acid) may be cleaved, exposing the next amino acid (e.g., n-1 amino acid) to the terminus (e.g., N-terminus). The methods described herein may be repeated. For example, upon removal of the terminal amino acids from the polypeptide and/or the additional polypeptide, the methods described herein may comprise detecting one or more signals or signal changes from the truncated (e.g., one amino acid less) polypeptide and/or the additional polypeptide (e.g., a modified polypeptide and/or the additional modified polypeptide). In some cases, upon removal of the terminal amino acids from the polypeptide and/or the additional polypeptide, the methods described herein my comprise detecting one or more signals or signal changes from the detection may use at least one fragment comprising the amino acid residue that has been cleaved from the polypeptide to identify the characteristic of the at least the portion of the polypeptide. The modified polypeptide and/or the additional modified polypeptide may comprise one fewer amino acid residue (e.g., two fewer amino acids, three fewer amino acids, or more) than prior to cleavage. In some cases, the methods described herein may further comprise detecting one or more signals or signal change (or no detectable signal or signal change when the probe is not associated with the terminal amino acids of the modified polypeptide and/or the additional modified polypeptide). For example, the next terminal amino acid may be coupled to a first probe (e.g., specifically bound to lysine) or a second probe (e.g., specifically bound to cysteine). In some cases, depending on the presence of the first probe or the second probe coupled to a terminal amino acid, one or more signal may be produced. In some cases, the one or more signals or signal change may be indicative of the presence and/or identity of the bound amino acid residue. Upon detection, the terminus (e.g., N-terminus or C- terminus) of the modified polypeptide and/or the additional modified polypeptide may be cleaved by exposing the modified polypeptide and/or the additional modified polypeptide to one or more conditions sufficient to cleave the terminal amino acid. In some cases, the one or more conditions may comprise the degradation agents described herein (e.g., the degradation agent comprising photo-cleavable moiety). In some cases, upon subjecting the one or more degradation agents coupled to the modified polypeptide and/or the additional modified polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N- terminal amino acid) may be cleaved, exposing the next amino acid (e.g., n-2 amino acid) to the terminus (e.g., N-terminus). In some cases, this process may be repeated iteratively, such that after each degradation event, a new signal corresponding to the next amino acid position (or the lack of the signal thereof) may be detected. In some cases, (1) the terminal amino acid e.g., N- terminal or C-terminal) removing step or operation (e.g., removal of the terminal amino acid using the degradation agent described herein), and/or (2) the detecting step or operation can be successively repeated from about 1 time to about 5 times, from about 5 times to about 10 times, from about 10 times to about 20 times, from about 20 times to about 30 times, from about 30 times to about 40 times, from about 40 times to about 50 times, from about 50 times to about 60 times, from about 60 times to about 70 times, from about 70 times to about 80 times, from about 80 times to about 90 times, or from about 90 times to about 100 times. In some cases, the methods described herein may be repeated at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, or more. In some cases, the methods described herein may be repeated at most about 5 times, at most about 10 times, at most about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times, or less. These operations and processes can be used to determine (1) at least one characteristic (or property) of at least a portion of the polypeptide and/or additional polypeptide and/or (2) at least one characteristic of a sample comprising the polypeptide and/or additional polypeptide.

[00189] In another aspect, provided herein are methods of detection at least one characteristic (e.g., sequence) of one or more samples or one or more polypeptides. In some cases, the methods may comprise providing one or more polypeptides (e.g., a polypeptide and/or an additional polypeptide). In some cases, the one or more polypeptides may be coupled to one or more probes (e.g., a probe and/or an additional probe). The probe and/or the additional probe may be coupled to N-terminal amino acids or C-terminal amino acids. Upon binding of the one or more probes to the terminal amino acids of polypeptide and/or the additional polypeptide, each probe-terminal amino acid conjugate may be cleaved from the polypeptide and/or the additional polypeptide using chemical, enzymatic, photolytic, thermal treatment, or any combination thereof, exposing the next amino acid (e.g., n-1 amino acid) to the terminus (e.g., N- terminus). In some cases, the removal of the probe-amino acid conjugate from the polypeptide and/or the additional polypeptide may be facilitated by subjecting the polypeptide and/or the additional polypeptide to one or more conditions sufficient to remove the one or more amino acid. For example, in some cases the one or more conditions may be a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, a degradation agent, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may include conjugating the polypeptide and/or the additional polypeptide may be coupled to a degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety). Upon cleavage of the probe-amino acid conjugate from the polypeptide and/or the additional polypeptide, the methods described herein can comprise detecting one or more signals or signal changes. When detection occurs after removing the terminal amino acids, the detection of signal or signal change can be of the cleaved off fragment comprising the terminal amino acid (coupled to one or more probes), and/or of the remaining shorter polypeptide (the modified polypeptide). Upon cleavage of the probe-amino acid conjugate, the modified polypeptide and/or the additional modified polypeptide comprising one amino acid fewer than the polypeptide and/or the modified polypeptide can be used for one or more additional cycles. For example, the methods may comprise coupling one or more probes (e.g., a probe and/or an additional probe) to N-terminal amino acids or C-terminal amino acids of the modified polypeptide and/or the additional modified polypeptide. Upon binding of the one or more probes to the terminal amino acids of modified polypeptide and/or the additional modified polypeptide, each probe-terminal amino acid conjugate may be cleaved from the polypeptide and/or the additional polypeptide using chemical, enzymatic, photolytic, thermal treatment, or any combination thereof, exposing the next amino acid (e.g., n-1 amino acid) to the terminus (e.g., N-terminus). In some cases, the removal of the probe-amino acid conjugate from the modified polypeptide or the additional modified polypeptide may be facilitated by the degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety). Upon cleavage of the probe-amino acid conjugate from the modified polypeptide and/or the additional modified polypeptide, the methods described herein can further comprise detecting one or more signals or signal changes. In some cases, (1) the conjugation step or operation of the polypeptide to one or more probes, (2) removal of the terminal amino acid of the polypeptide and/or the additional polypeptide using the degradation agent described herein), and/or (4) the detecting step or operation can be successively repeated from about 1 time to about 5 times, from about 5 times to about 10 times, from about 10 times to about 20 times, from about 20 times to about 30 times, from about 30 times to about 40 times, from about 40 times to about 50 times, from about 50 times to about 60 times, from about 60 times to about 70 times, from about 70 times to about 80 times, from about 80 times to about 90 times, or from about 90 times to about 100 times. In some cases, the methods described herein may be repeated at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, or more. In some cases, the methods described herein may be repeated at most about 5 times, at most about 10 times, at most about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times, or less. These operations and processes can be used to determine (1) at least one characteristic (or property) of at least a portion of the polypeptide and/or additional polypeptide and/or (2) at least one characteristic of a sample comprising the polypeptide and/or additional polypeptide.

[00190] In another aspect, provided herein are methods of detection at least one characteristic (e.g., sequence) of one or more samples and/or one or more polypeptides. In some cases, the methods may comprise providing one or more polypeptides (e.g., a polypeptide and an additional polypeptide). In some cases, the one or more polypeptides may be coupled to one or more probes (e.g., a probe or an additional probe). In some cases, the probe or the additional probe may be coupled to specific amino acids. For example, the probe may be coupled to any lysine residues (e.g., N-terminal lysine residues, C-terminal lysine residues, and/or internal lysine residues) of the polypeptide and/or the additional polypeptide, while the additional probe may be coupled to any cysteine residues (e.g., N-terminal cysteine residues, C-terminal cysteine residues, and/or internal cysteine residues) of the polypeptide and/or the additional polypeptide. In some cases, the one or more probes, when bound to one or more amino acids of the polypeptide or the additional polypeptide may be configured to produce one or more signals or signal changes. In some cases, the terminus (e.g., N-terminus or C-terminus) of the polypeptide and/or the additional polypeptide may be cleaved when subjected to one or more conditions sufficient to remove the one or more amino acid. In some cases, the one or more conditions sufficient to remove the one or more amino acid from the polypeptide or the additional polypeptide maybe cleaved upon subjecting the polypeptide and/or the additional polypeptide to one or more conditions sufficient to cleave the one or more amino acids. For example, in some cases the one or more conditions may be a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, a degradation agent, water solubility of the degradation agent, or any combination thereof. In some cases, the one or more conditions may include conjugating the polypeptide and/or the additional polypeptide may be coupled to a degradation agent described herein (e.g., a degradation agent comprising a photo- cleavable moiety). In some cases, upon subjecting the one or more degradation agents coupled to the polypeptide and/or the additional polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N-terminal amino acid) may be cleaved, exposing the next amino acid (e.g., n-1 amino acid) to the terminus (e.g., N-terminus). Depending on the specific amion residue, the next terminal amino acid may or may not be coupled to one or more probes described herein. Upon removal of the terminal amino acids from the polypeptide and/or the additional polypeptide, the methods described herein may comprise detecting one or more signals or signal changes. In some cases, upon removal of the terminal amino acids from the polypeptide and/or the additional polypeptide, the methods described herein my comprise detecting one or more signals or signal changes from the at least one fragment comprising the amino acid residue that has been cleaved from the polypeptide. In some cases, the detection may comprise detecting one or more signals or signal change of the modified polypeptide or the additional polypeptide. Upon detection, in some cases, the methods described herein may further comprise subjecting the modified polypeptide and/or the additional modified polypeptide to one or more conditions sufficient to remove the terminal amino acid. In some cases, the one or more conditions sufficient to remove the terminal amino acid may comprise conjugating (coupling) a terminal amino acid of the modified polypeptide and/or the additional modified polypeptide to a degradation agents described herein (e.g., the degradation agent comprising photo-cleavable moiety). In some cases, upon subjecting the one or more degradation agents coupled to the modified polypeptide and/or the additional modified polypeptides to light comprising one or more wavelengths, the terminal amino acid (e.g., N-terminal amino acid) may be cleaved, exposing the next amino acid (e.g., n-2 amino acid) to the terminus (e.g., N-terminus). Upon removal of the terminal amino acids from the modified polypeptide and/or the additional modified polypeptide, the methods described herein may comprise detecting one or more signals or signal changes. In some cases, upon removal of the terminal amino acids from the polypeptide and/or the additional polypeptide, the methods described herein my comprise detecting one or more signals or signal changes from the at least one fragment comprising the amino acid residue that has been cleaved from the polypeptide to identify the characteristic of the at least the portion of the polypeptide. In some cases, the detection may comprise detecting one or more signals or signal changes from the next terminal amino acids exposed from the first modified polypeptide and the additional modified polypeptide. In some cases, this process may be repeated iteratively, such that after each degradation event, a new signal corresponding to the next amino acid position (or the lack of the signal thereof) may be detected. In some cases, this process may be repeated iteratively, such that after each degradation event, a new signal corresponding to the cleaved amino acid (that are bound to one or more probes described herein) may be detected. In some cases, (1) the terminal amino acid (e.g., N-terminal or C-terminal) removing step or operation (e.g., removal of the terminal amino acid using the degradation agent described herein), and/or (2) the detecting step or operation can be successively repeated from about 1 time to about 5 times, from about 5 times to about 10 times, from about 10 times to about 20 times, from about 20 times to about 30 times, from about 30 times to about 40 times, from about 40 times to about 50 times, from about 50 times to about 60 times, from about 60 times to about 70 times, from about 70 times to about 80 times, from about 80 times to about 90 times, or from about 90 times to about 100 times. In some cases, the methods described herein may be repeated at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, or more. In some cases, the methods described herein may be repeated at most about 5 times, at most about 10 times, at most about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times, or less. These operations and processes can be used to determine (1) at least one characteristic (or property) of at least a portion of the polypeptide and/or additional polypeptide and/or (2) at least one characteristic of a sample comprising the polypeptide and/or additional polypeptide.

[00191] In another aspect, provided herein are methods for analyzing one or more samples. In some cases, the polypeptide and at least one additional polypeptide may be provided. In some cases, the polypeptide and the at least one additional polypeptide may be from the same sample. In some cases, the polypeptide and the at least one additional polypeptide may be from different samples. In some cases, the polypeptide may be provided to a first location on a first support. The at least one additional polypeptide may be provided to an additional location on an additional support. In some cases, the methods described herein may comprise providing a sample comprising a peptide and an additional peptide. In some cases, one or more probes may be coupled to one or more amino acids of the peptide. In some cases, one or more additional probes may be coupled to one or more amino acids of the additional peptide. In some cases, the one or more probes and/or the one or more additional probes may be coupled to the same amino acid or the same amino acid type (e.g., N-terminal amino acids). In some cases, the one or more probes or the one or more additional probes may be coupled to specific amino acids. For example, the one or more probes may be coupled to any lysine residues (e.g., N-terminal lysine residues, C-terminal lysine residues, and/or internal lysine residues) of the polypeptide and/or the additional polypeptide, while the one or more additional probes may be coupled to any cysteine residues (e.g., N-terminal cysteine residues, C-terminal cysteine residues, and/or internal cysteine residues) of the polypeptide and/or the additional polypeptide. In some cases, the methods described herein may comprise detecting one or more signals or signal change from the one or more probes and/or one or more signals or signal change from the one or more additional probes. In some cases, a terminus (e.g., each terminus) of the polypeptide and at least one additional polypeptide may be contacted with a degradation agent and/or an additional degradation agent. For example, in some cases, the terminus of the polypeptide may be contacted with a degradation agent (e.g., a degradation agent comprising a photo-cleavable moiety), and/or the terminus of the additional polypeptide may be contacted with an additional degradation agent. Upon contacting with the degradation agent or the additional degradation agents, the terminus (e.g., each terminus of the polypeptide and the additional polypeptides) may be removed. For example, in some cases, the terminal amino acids of the polypeptide and/or the terminal amino acid of the additional polypeptide may be removed upon subjecting the polypeptide and/or the additional polypeptide (e.g., that are coupled to the degradation agent or the additional degradation agent) to light comprising one or more wavelengths. In some cases, the methods further comprise identifying one or more characteristics of the sample. In some cases, the one or more characteristics of the sample may be determined by detecting one or more signals or signal changes that are unique to a specific amino acid that are bound to one or more probes described herein.

[00192] In some cases, the methods may comprise providing a sample e.g., a biological sample) comprising a first polypeptide and a second polypeptide. In some cases, the first polypeptide may be coupled to a first one or more probes. In some cases, the second polypeptide is coupled to a second one or more probes. In some cases, the first one or more probes and the second one or more probes may be the same. For example, in some cases, the first one or more probes and the second one or more probes may be the degradation agent comprising a photocleavable moiety. In other cases, the first one or more probes and the second one or more probes may be an amino acid specific probe (e.g., a probe that binds to lysine). In some cases, the first one or more probes and the second one or more probes may be different. For example, in some cases, the first one or more probes may be coupled to a specific amino acid of the first polypeptide. In some cases, the second one or more probes may be coupled to a specific amino acid (that is different target of the first one or more probes) of the second polypeptide. In some cases, the methods may further comprise detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes. In some cases, the method may further comprise contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent, thereby removing at least one amino acid from the first polypeptide and/or from the second polypeptide. In some cases, the first degradation agent may comprise a first photo-cleavable moiety and/or the second degradation agent may comprise a second photo- cleavable moiety. In some cases, the method may further comprise identifying one or more characteristics of the sample.

[00193] In another aspect, methods described herein may comprise providing a polypeptide. In some cases, the polypeptide may comprise one or more amino acids coupled to one or more probes. In some cases, the one or more probes may be configured to bind to a specific amino acid (of the polypeptide), an amino acid type (of the polypeptide), N-terminal amino acid, C- terminal amino acids, or internal amino acids. In some cases, the one or more probes may be bound to one or more natural amino acids. In some cases, the one or more probes may bind to one or more unnatural amino acids. As used herein, the term “natural amino acid” may refer to any of the twenty standard a-amino acids (e.g., Table 2) that are genetically encoded and incorporated into proteins. As used herein, the term “unnatural amino acid” (also referred to as ‘non-natural amino acids’) may refer to any amino acids that are not among the twenty genetically encoded amino acids. For example, unnatural amino acids may include chemically modified analogs of natural amino acids, amino acids with altered side chains or backbone structures, or synthetically derived amino acids not found in nature. Examples include, but are not limited to, p-azido-L-phenylalanine, norleucine, or post-translationally modified amino acids (e.g., phosphorylated or methylated).

[00194] As used herein, the term “specific amino acid” may refer to an individual, identifiable amino acid selected from among the twenty genetically encoded amino acids or any chemically synthesized amino acid, designated by its common name, three-letter code, or one- letter code. Examples include arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), or valine (Vai).

[00195] As used herein, the term “amino acid type” may refer to a classification of amino acids based on shared physicochemical properties, such as polarity, charges, or hydrophobicity. In some cases, the term may refer to a classification of amino acids based on positional context within a polypeptide or protein. For example, physicochemical classifications may include hydrophobic amino acids (e.g., leucine, isoleucine, valine), polar uncharged amino acids (e.g., serine, threonine), acidic amino acids (e.g., aspartic acid, glutamic acids), basic amino acids (e.g., lysine, arginine), and aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine). In some cases, positional classification may include amino acids located at or near the N-terminus or C-terminus of a polypeptide, internal amino acids, or amino acids located within a defined motif or domain.

[00196] In some cases, upon binding of the one or more probes to one or more amino acids of the polypeptide, the one or more probes may produce one or more signals or signal change (e.g., in response to light). In some cases, the one or more signals or signal changes may be unique to the one or more amino acids bound to the one or more probe. In some cases, the method may further comprise detecting one or more signals or signal changes from the one or more probes on the polypeptide. In some cases, the one or more signals or signal change may be used to determine at least one characteristic of the at least a portion of the polypeptide (e.g., sequencing). In some cases, the methods may further comprise subjecting at least a portion of the polypeptide to a first light comprising a wavelength from 200 nm to 750 nm. In some cases, the subjecting at least a portion of the polypeptide may initiate photodegradation of the one or more probes that are conjugated to one or more amino acids (of the polypeptide), thereby leaving the original one or more amino acids. In some cases, the methods may further comprise contacting one or more amino acids of the polypeptide with one or more degradation agents comprising a photo-cleavable moiety described herein. In some cases, upon subjecting at least a portion of the polypeptide to a second light comprising a wavelength, a terminal amino acid of the polypeptide may be removed.

[00197] In some cases, the methods, systems, compositions, and/or kits described herein may comprise detecting one or more signals or signal changes. For example, in some cases, one or more probes coupled to one or more amino acids of a peptide or an additional peptide may be configured to produce one or more signals or signal change. In some cases, the one or more signals or signal change may be unique to the one or more amino acids that the one or more probes are bound to. In some cases, the one or more signals or signal change may be used to determine at least one characteristic of the at least a portion of the polypeptide or the additional polypeptide. For example, in some cases, the at least one characteristic may comprise a number of polypeptides in the sample, type of polypeptides in the sample, an origin of the sample, impurities in the sample, presence of a polypeptide, absence of a polypeptide, or any combination thereof. In some cases, the at least one characteristic may be determined by using sequence information determined by the methods described herein. In some cases, upon excitation with light of an appropriate wavelength, the one or more probes may emit signals characterized by one or more emission properties, including but not limited to fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof. These emission properties may be measured and analyzed to generate a signal profile corresponding to the specific residue or sequence context. The measured emission profile may then be compared to one or more reference values or profiles, which may be derived from known standards, calibration libraries, or previously characterized sequences. Based on the comparison, the identity or position of the amino acid residue(s) may be determined. In some cases, multiple emission properties may be used in combination to improve specificity and/or accuracy of residue identification.

Peptide Degradation

[00198] Provided herein are methods, systems, compositions, and/or kits for polypeptide degradation using a range of chemical and enzymatic techniques or a degradation agent described herein. In some cases, the degradation may be sequential polypeptide degradation. A polypeptide may be iteratively subjected to cleavage conditions to determine one or more characteristics (e.g., sequence) of at least a portion of the polypeptide. Controlled amino acid removal (e.g., N- or C-terminal amino acid removal) may be carried out through a variety of techniques including, for example, degradation, organophosphate degradation, or proteolytic cleavage. In some instances, the N-terminal amino acid residue can be selectively removed from a polypeptide. In some instances, the C-terminal amino acid residue can be selectively removed from a polypeptide. A chemical or enzymatic technique for removing a terminal amino acid may remove a defined number of (e.g., at least one, at least two, at least three or more) amino acids. Accordingly, a method for analyzing a polypeptide may comprise successive degradation and analysis step or operations, such that the removal of a defined number of amino acids from an N-terminus or C-terminus per step or operation provides position and sequence specific amino acid identifications during analysis. A chemical or enzymatic technique for removing a terminal amino acid may cleave a polypeptide at a defined location (e.g., only in between two alanine residues, or only at the polypeptide bond connecting an N- terminal amino acid to the remainder of a polypeptide).

[00199] Provided herein are methods, systems, composition, and/or kit described herein for degrading one or more amino acids (e.g., internal, C-terminus, and/or N-terminus amino acids) from an analyte (e.g., a polypeptide). In some cases, the degradation of one or more amino acids may comprise using Edman, or related, chemical degradation. Alternatively, the degradation of one or more amino acids may comprise enzymatic degradation with a protease, such as an aminopeptidase or carboxypeptidase. In other cases, the degradation of one or more amino acids may comprise a degradation agent described herein. In some cases, the degradation agent described herein may degrade (e.g., cleave) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acids. In some cases, the degradation agent described herein may degrade (e.g., cleave) at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten, or less amino acids.

[00200] Provided herein are methods, systems, compositions, and/or kits disclosed herein for degrading one or more amino acids (e.g., internal, C-terminus, and/or N-terminus amino acids) from an analyte (e.g., a polypeptide) using a degradation agent described herein (e.g., 3.45). [00201] In some cases, the degradation agent described herein can provide a controlled degradation step or operations by regulating (1) conjugation step or operation (e.g., guanidinylation of the terminal amino acid) and/or (2) degradation (e.g., cleaving of an amino acid) step or operation, thereby minimizing (e.g., preventing) iterative (e.g., at least about 2 times, at least about 3 times, at least about 5 times, at least about 10 times or more), uninhibited removal of the one or more amino acids (e.g., N-terminal amino acids). For example, the degradation agent provided herein may provide spatial and temporal control of degradation (e.g., cleavage) by decoupling the conjugation step or operation (by conjugating to the degradation agent to one or more amino acid) and the degradation (e.g., cleavage step or operation). Chemically triggered N-degradation methods cannot control which proteins are degraded in space of an immobilized protein analyte. This is due to the fact that the chemical trigger may be applied to the whole area or volume of study. However, the degradation agent described herein may use light as a trigger which means that only the area that is irradiated with light will be degraded and the remaining area will be unaffected and undegraded. For example, in some cases, light can be applied to at least a portion of the first polypeptide and not the second polypeptide, thereby resulting in degradation of one or more amino acids from the first polypeptide, but not the second polypeptide. [00202] In some cases, the methods described herein may comprise contacting at least a portion of the polypeptide with a degradation agent, thereby forming the polypeptide coupled to a degradation agent (e.g., a first modified polypeptide). In some cases, the first modified polypeptide may comprise a photo-cleavable moiety. In some cases, the methods may comprise subjecting the at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide. In some cases, the second modified polypeptide may have one or more fewer amino acids than the polypeptide. In some cases, the condition sufficient to generate a second modified polypeptide may include a pH condition, an optical condition, a duration condition, a buffer condition, temperature, water solubility, or any combination thereof of the degradation agent.

[00203] In some cases, the methods described herein may comprise contacting at least a portion of a polypeptide and/or at least a portion of an additional polypeptide with one or more degradation agents. In some cases, the one or more degradation that may be coupled to the at least a portion of the polypeptide and/or at least a portion of the additional polypeptide may be the same (e.g., a degradation agent comprising a photo-cleavable moiety). In other cases, the one or more degradation that may be coupled to the at least a portion of the polypeptide and/or at least a portion of the additional polypeptide may be different. For example, in some cases, the at least a portion of the peptide may be coupled to a degradation agent, while the at least a portion of the additional peptide may be coupled to an additional degradation agent. In some cases, the degradation agent may be a degradation agent comprising a photo-cleavable moiety, while the additional degradation agent may be an additional degradation agent comprising an Edman degradation agent (e.g., phenylisothiocyanate or PITC), l-fluoro-2, 4, dinitrobenzene (FDNB), dansyl chloride, hydrazinolysis, and/or enzymatic and chemical cleavage agents (e.g., cyanogen bromide, trypsin, chymotrypsin, pepsin, or thermolysin). In some cases, the degradation agent and the additional degradation agent may be coupled to cleave one or more amino acids (e.g., terminal amino acids) in response to one or more conditions. In some cases, the one or more conditions may comprise a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof. For example, in some cases, the degradation agent may cleave one or more amino acids (e.g., N-terminal amino acids) in response to a first condition, while the additional degradation agent may cleave one or more amino acids in response to a second condition. In some cases, the methods described herein may comprise subjecting a polypeptide coupled to a degradation agent and the additional polypeptide coupled to an additional degradation agent to a first condition (e.g., light), thereby facilitating the removal of one or more amino acids of the polypeptide, but leaving the one or more amino acids of the additional polypeptide intact (e.g., no removal of one or more amino acids from the additional polypeptide).

[00204] A condition sufficient to generate a modified polypeptide (e.g., a first modified polypeptide and/or a second modified polypeptide) may include buffer condition. In some cases, the degradation agent described herein may bind to the one or more amino acids polypeptide and/or degrade (e.g., cleave) the one or more amino acids from the polypeptide under basic aqueous conditions. For example, the degradation agent can bind to the one or more amino acids of polypeptide and/or degrade (e.g., cleave) the one or more amino acids from the polypeptide in a basic aqueous solution lacking or substantially lacking an organic solvent or a harsh chemical (e.g., TFA for removing the protecting group). For example, the methods, systems, compositions, and/or kits described herein can result in at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more degradation of the one or more amino acids from the polypeptide under basic aqueous conditions lacking or substantially lacking an organic solvent or a harsh chemicals. In some cases, the conjugation step or operation (e.g., binding of a degradation agent to the one or more amino acids of polypeptide) and the degradation step or operation (e.g., cleaving) can occur in the same aqueous buffer (e.g., basic aqueous buffer), minimizing the need for dangerous chemicals or reagents. The degradation agent may comprise one or more aromatic groups and/or comprises a nitro-substituted benzyl group.

[00205] In some cases, the degradation agent described herein (e.g., 3.45) may be conjugated to one or more amino acids (e.g., N-terminus, C-terminus or internal amino acids) of a polypeptide. In some cases, the polypeptide may be in a solution (e.g., basic aqueous buffer). In some cases, the polypeptide may be bound to a surface or support as described herein. In some cases, the conjugation of the degradation agent to the one or more amino acids may result in guanidinylation of the one or more amino acids. In some cases, the basic aqueous buffer lacks or substantially lacks an organic solvent or cosolvent, such as, TFA, boron trifluoride dietherate, and/or pyridine. For example, in some cases, the basic aqueous buffer may comprise at most about 15%, at most about 10%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.5%, or less of an organic solvent or cosolvent. [00206] In some cases, the buffer may be a basic aqueous buffer. In some cases, the buffer may be a bicarbonate buffer, an ammonia buffer, a tris buffer, a borate buffer, or any combination thereof. In some cases, the basic buffer may be a bicarbonate buffer. In some cases, a concentration of the buffer is from about 0.2 mM to about 5 mM. In some cases, the concentration of the buffer is about 0.2 mM to about 0.4 mM, from about 0.2 mM to about 0.6 mM, from about 0.2 mM to about 0.8 mM, from about 0.2 mM to about 1 mM, from about 0.2 mM to about 1.5 mM, from about 0.2 mM to about 2 mM, from about 0.2 mM to about 2.5 mM, from about 0.2 mM to about 3 mM, from about 0.2 mM to about 3.5 mM, from about 0.2 mM to about 4 mM, from about 0.2 mM to about 5 mM, from about 0.4 mM to about 0.6 mM, from about 0.4 mM to about 0.8 mM, from about 0.4 mM to about 1 mM, from about 0.4 mM to about 1.5 mM, from about 0.4 mM to about 2 mM, from about 0.4 mM to about 2.5 mM, from about 0.4 mM to about 3 mM, from about 0.4 mM to about 3.5 mM, from about 0.4 mM to about 4 mM, from about 0.4 mM to about 5 mM, from about 0.6 mM to about 0.8 mM, from about 0.6 mM to about 1 mM, from about 0.6 mM to about 1.5 mM, from about 0.6 mM to about 2 mM, from about 0.6 mM to about 2.5 mM, from about 0.6 mM to about 3 mM, from about 0.6 mM to about 3.5 mM, from about 0.6 mM to about 4 mM, from about 0.6 mM to about 5 mM, from about 0.8 mM to about 1 mM, from about 0.8 mM to about 1.5 mM, from about 0.8 mM to about 2 mM, from about 0.8 mM to about 2.5 mM, from about 0.8 mM to about 3 mM, from about 0.8 mM to about 3.5 mM, from about 0.8 mM to about 4 mM, from about 0.8 mM to about 5 mM, from about 1 mM to about 1.5 mM, from about 1 mM to about 2 mM, from about 1 mM to about 2.5 mM, from about 1 mM to about 3 mM, from about 1 mM to about 3.5 mM, from about 1 mM to about 4 mM, from about 1 mM to about 5 mM, from about 1.5 mM to about 2 mM, from about 1.5 mM to about 2.5 mM, from about 1.5 mM to about 3 mM, from about 1.5 mM to about 3.5 mM, from about 1.5 mM to about 4 mM, from about 1.5 mM to about 5 mM, from about 2 mM to about 2.5 mM, from about 2 mM to about 3 mM, from about 2 mM to about 3.5 mM, from about 2 mM to about 4 mM, from about 2 mM to about 5 mM, from about 2.5 mM to about 3 mM, from about 2.5 mM to about 3.5 mM, from about 2.5 mM to about 4 mM, from about 2.5 mM to about 5 mM, from about 3 mM to about 3.5 mM, from about 3 mM to about 4 mM, from about 3 mM to about 5 mM, from about 3.5 mM to about 4 mM, from about 3.5 mM to about 5 mM, or from about 4 mM to about 5 mM. In some cases, the concentration of the buffer is at least about 0.2 mM, at least about 0.4 mM, at least about 0.6 mM, at least about 0.8 mM, at least about 1 mM, at least about 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about 3 mM, at least about 3.5 mM, at least about 4 mM, more. In some cases, the concentration of the buffer is at most about 0.4 mM, at most about 0.6 mM, at most about 0.8 mM, at most about 1 mM, at most about 1.5 mM, at most about 2 mM, at most about 2.5 mM, at most about 3 mM, at most about 3.5 mM, at most about 4 mM, at most about 5 mM, or less. [00207] In some cases, the degradation agent described herein can readily bind to the polypeptide (e.g., conjugated to N-terminal amino acid, C-terminal amino acids, or internal amino acids) in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents. In some cases, the basic aqueous buffer described herein can lead to minimal (e.g., no) polypeptide degradation (e.g., polypeptide bond hydrolysis, denaturation, or side chain modifications). In some cases, pH of the aqueous buffer described here can be at pH about from 1 to about 14. In some cases, pH of the aqueous buffer described here can be at pH from about 1 to about 3, from about 1 to about 5, from about 1 to about 6, from about 1 to about 7, from about 1 to about 8, from about 1 to about 9, from about 1 to about 10, from about 1 to about 11, from about 1 to about 12, from about 1 to about 13, from about 1 to about 14, from about 3 to about 5, from about 3 to about 6, from about 3 to about 7, from about 3 to about 8, from about 3 to about 9, from about 3 to about 10, from about 3 to about 11, from about 3 to about 12, from about 3 to about 13, from about 3 to about 14, from about 5 to about 6, from about 5 to about 7, from about 5 to about 8, from about 5 to about 9, from about 5 to about 10, from about 5 to about 11, from about 5 to about 12, from about 5 to about 13, from about 5 to about 14, from about 6 to about 7, from about 6 to about 8, from about 6 to about 9, from about 6 to about 10, from about 6 to about 11, from about 6 to about 12, from about 6 to about 13, from about 6 to about 14, from about 7 to about 8, from about 7 to about 9, from about 7 to about 10, from about 7 to about 11, from about 7 to about 12, from about 7 to about 13, from about 7 to about 14, from about 8 to about 9, from about 8 to about 10, from about 8 to about 11, from about 8 to about 12, from about 8 to about 13, from about 8 to about 14, from about 9 to about 10, from about 9 to about 11, from about 9 to about 12, from about 9 to about 13, from about 9 to about 14, from about 10 to about 11, from about 10 to about 12, from about 10 to about 13, from about 10 to about 14, from about 11 to about 12, from about 11 to about 13, from about 11 to about 14, from about 12 to about 13, from about 12 to about 14, or from about 13 to about 14. In some cases, pH of the basic aqueous buffer described here can be at pH at least about 1, at least about 3, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, about 13, or more. In some cases, pH of the basic aqueous buffer described here can be at pH at most about 3, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14 or less.

[00208] In some cases, the degradation reaction (e.g., cleaving one or more amino acids) may be accelerated in the presence of Lewis acids in a reaction solution as compared to a control reaction conducted under identical conditions but lacking Lewis acids. In some cases, a solution comprising Lewis acid may comprise scandium triflate, ytterbium tritiate and/or zinc tritiate. In some cases, the degradation reaction may be accelerated by at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 1.5 hours, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours or more as compared to a control reaction conducted under identical conditions but lacking Lewis acids. In some cases, the Lewis acids can be water soluble. In some cases, the Lewis acids may coordinate with the amide carbonyl oxygen, thereby making it more electrophilic. Non-limiting examples of Lewis acids may include lithium (Li), ytterbium (Yb), or scandium (SC). In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted in a buffer having pH of from about 7 to about 13. In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted in a buffer having pH of from about 7 to about 8, from about 7 to about 9, from about 7 to about 10, from about 7 to about 11, from about 7 to about 12, from about 7 to about 13, from about 8 to about 9, from about 8 to about 10, from about 8 to about 11, from about 8 to about 12, from about 8 to about 13, from about 9 to about 10, from about 9 to about 11, from about 9 to about 12, from about 9 to about 13, from about 10 to about 11, from about 10 to about 12, from about 10 to about 13, from about 11 to about 12, from about 11 to about 13, or about 12 to about 13. In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted in a buffer having pH of at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, about 12, or more. In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted in a buffer having pH of at most about 8, at most about 9, at most about 10, at most about 11, at most about 12, about 13 or less.

[00209] In some cases, the concentration of the Lewis acid in the reaction solution is from about 0.1 M to about 1 M. The concentration of the Lewis acid in the reaction solution is about 0.1 M to about 0.2 M, from about 0.1 M to about 0.3 M, from about 0.1 M to about 0.4 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 0.6 M, from about 0.1 M to about 0.7 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.9 M, from about 0.1 M to about 1 M, from about 0.2 M to about 0.3 M, from about 0.2 M to about 0.4 M, from about 0.2 M to about 0.5 M, from about 0.2 M to about 0.6 M, from about 0.2 M to about 0.7 M, from about 0.2 M to about 0.8 M, from about 0.2 M to about 0.9 M, from about 0.2 M to about 1 M, from about 0.3 M to about 0.4 M, from about 0.3 M to about 0.5 M, from about 0.3 M to about 0.6 M, from about 0.3 M to about 0.7 M, from about 0.3 M to about 0.8 M, from about 0.3 M to about 0.9 M, from about 0.3 M to about 1 M, from about 0.4 M to about 0.5 M, from about 0.4 M to about 0.6 M, from about 0.4 M to about 0.7 M, from about 0.4 M to about 0.8 M, from about 0.4 M to about 0.9 M, from about 0.4 M to about 1 M, from about 0.5 M to about 0.6 M, from about 0.5 M to about 0.7 M, from about 0.5 M to about 0.8 M, from about 0.5 M to about 0.9 M, from about 0.5 M to about 1 M, from about 0.6 M to about 0.7 M, from about 0.6 M to about 0.8 M, from about 0.6 M to about 0.9 M, from about 0.6 M to about 1 M, from about 0.7 M to about 0.8 M, from about 0.7 M to about 0.9 M, from about 0.7 M to about 1 M, from about 0.8 M to about 0.9 M, from about 0.8 M to about 1 M, or about 0.9 M to about 1 M. The concentration of the Lewis acid in the reaction solution is at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, or more. The concentration of the Lewis acid in the reaction solution is at most about 0.2 M, at most about 0.3 M, at most about 0.4 M, at most about 0.5 M, at most about 0.6 M, at most about 0.7 M, at most about 0.8 M, at most about 0.9 M, at most about 1 M or less.

[00210] In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted at temperature from about 25 °C to about 60 °C. In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted at temperature from about 25 °C to about 30 °C, from about 25 °C to about 35 °C, from about 25 °C to about 40 °C, from about 25 °C to about 45 °C, from about 25 °C to about 50 °C, from about 25 °C to about 55 °C, from about 25 °C to about 60 °C, from about 30 °C to about 35 °C, from about 30 °C to about 40 °C, from about 30 °C to about 45 °C, from about 30 °C to about 50 °C, from about 30 °C to about 55 °C, from about 30 °C to about 60 °C, from about 35 °C to about 40 °C, from about 35 °C to about 45 °C, from about 35 °C to about 50 °C, from about 35 °C to about 55 °C, from about 35 °C to about 60 °C, from about 40 °C to about 45 °C, from about 40 °C to about 50 °C, from about 40 °C to about 55 °C, from about 40 °C to about 60 °C, from about 45 °C to about 50 °C, from about 45 °C to about 55 °C, from about 45 °C to about 60 °C, from about 50 °C to about 55 °C, from about 50 °C to about 60 °C, or about 55 °C to about 60 °C. In some cases, the degradation reaction (e.g., in the presence of Lewis acids) may be conducted at temperature about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, or about 60 °C. In some cases, the degradation reaction (e.g, in the presence of Lewis acids) may be conducted at temperature at least about 25 °C, at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, about 55 °C, or more. In some cases, the degradation reaction (e.g, in the presence of Lewis acids) may be conducted at temperature at most about 30 °C, at most about 35 °C, at most about 40 °C, at most about 45 °C, at most about 50 °C, at most about 55 °C, about 60 °C or more. [00211] In some cases, a condition sufficient to generate a modified polypeptide (e.g., a first modified polypeptide and/or a second modified polypeptide) may include water solubility of the degradation agent. In some cases, the water solubility of the degradation agent can be measured using partition coefficient value (logP). In some cases, the logP value of the degradation agent (e.g., water solubility of the degradation agent) in a phase (e.g., in an aqueous environment such as water and/or organic environment such as octanol) can be from -5 to 10. In some cases, the logP value of the degradation agent (e.g., water solubility of the degradation agent) in a phase (e.g., in an aqueous environment such as water and/or organic environment such as octanol) can be from about -5 to about -3, about from -5 to about -2, about from -5 to about -1, about from -5 to about 0, about from -5 to about 1, about from -5 to about 2, about from -5 to about 3.5, about from -5 to about 4, about from -5 to about 6, about from -5 to about 8, about from -5 to about 10, about from -3 to about -2, about from -3 to about -1, about from -3 to about 0, about from -3 to about 1, about from -3 to about 2, about from -3 to about 3.5, about from -3 to about 4, about from -3 to about 6, about from -3 to about 8, about from -3 to about 10, about from -2 to about - 1, about from -2 to about 0, about from -2 to about 1, about from -2 to about 2, about from -2 to about 3.5, about from -2 to about 4, about from -2 to about 6, about from -2 to about 8, about from -2 to about 10, about from -1 to about 0, about from -1 to about 1, about from -1 to about 2, about from -1 to about 3.5, about from -1 to about 4, about from -1 to about 6, about from -1 to about 8, about from -1 to about 10, about from 0 to about 1, about from 0 to about 2, about from 0 to about 3.5, about from 0 to about 4, about from 0 to about 6, about from 0 to about 8, about from 0 to about 10, about from 1 to about 2, about from 1 to about 3.5, about from 1 to about 4, about from 1 to about 6, about from 1 to about 8, about from 1 to about 10, about from 2 to about 3.5, about from 2 to about 4, about from 2 to about 6, about from 2 to about 8, about from 2 to about 10, about from 3.5 to about 4, about from 3.5 to about 6, about from 3.5 to about 8, about from 3.5 to about 10, about from 4 to about 6, about from 4 to about 8, about from 4 to about 10, about from 6 to about 8, about from 6 to about 10, or about 8 to about 10. In some cases, the logP value of the degradation agent (e.g., water solubility of the degradation agent) in a phase (e.g., in an aqueous environment such as water and/or organic environment such as octanol) can be at least about -5, at least about -3, at least about -2, at least about -1, at least about 0, at least about 1, at least about 2, at least about 3.5, at least about 4, at least about 6, at least about 8, or more. In some cases, the logP value of the degradation agent (e.g., water solubility of the degradation agent) in a phase (e.g., in an aqueous environment such as water and/or organic environment such as octanol) can be at most about -3, at most about -2, at most about -1, at most about 0, at most about 1, at most about 2, at most about 3.5, at most about 4, at most about 6, at most about 8, at most about 10, or less.

[00212] In some cases, the degradation agent can bind to the polypeptide (e.g., conjugated to N-terminal amino acid, C-terminal amino acids, or internal amino acids) within about 5 minutes to about 40 minutes in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents. The degradation agent can bind to the polypeptide (e.g., conjugated to N-terminal amino acid, C-terminal amino acids, or internal amino acids) from about 5 minutes to about 10 minutes, from about 5 minutes to about 15 minutes, from about 5 minutes to about 20 minutes, from about 5 minutes to about 25 minutes, from about 5 minutes to about 30 minutes, from about 5 minutes to about 35 minutes, from about 5 minutes to about 40 minutes, from about 10 minutes to about 15 minutes, from about 10 minutes to about 20 minutes, from about 10 minutes to about 25 minutes, from about 10 minutes to about 30 minutes, from about 10 minutes to about 35 minutes, from about 10 minutes to about 40 minutes, from about 15 minutes to about 20 minutes, from about 15 minutes to about 25 minutes, from about 15 minutes to about 30 minutes, from about 15 minutes to about 35 minutes, from about 15 minutes to about 40 minutes, from about 20 minutes to about 25 minutes, from about 20 minutes to about 30 minutes, from about 20 minutes to about 35 minutes, from about 20 minutes to about 40 minutes, from about 25 minutes to about 30 minutes, from about 25 minutes to about 35 minutes, from about 25 minutes to about 40 minutes, from about 30 minutes to about 35 minutes, from about 30 minutes to about 40 minutes, or from about 35 minutes to about 40 minutes in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents. The degradation agent can bind to the polypeptide (e.g., conjugated to N-terminal amino acid, C-terminal amino acids, or internal amino acids) within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents. The degradation agent can bind to the polypeptide (e.g., conjugated to N- terminal amino acid, C-terminal amino acids, or internal amino acids) in at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, or at least about 35 minutes or more in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents. The degradation agent can be bind to the polypeptide (e.g., conjugated to N-terminal amino acid, C- terminal amino acids, or internal amino acids) by at most about 10 minutes, at most about 15 minutes, at most about 20 minutes, at most about 25 minutes, at most about 30 minutes, at most about 35 minutes, at most about 40 minutes or less in basic aqueous buffer that lacks or substantially lacks an organic solvent or cosolvents.

[00213] In some cases, the degradation agent provided herein may be a photocaged degrader agent configured to provide trigger-and-release cleavage mechanism. For example, in some cases, the degradation agent that is bound to the polypeptide (e.g., upon formation of the one or more guanidylated amino acid) may be inert prior to photo-decaging (e.g., irradiation). The ability to control the degradation reaction (e.g., via trigger-and-release cleavage mechanism) may provide a significant advantage over other degradation methods (e.g., Edman degradation, and/or enzymatic/chemical degradation) in which the reaction proceeds uncontrollable. In particular, controlled degradation enables improved reproducibility, temporal precision, and tunability of the reaction. For example, in some cases, the unreacted degradation agents may be washed away in the dark, to control degradation agents. In some cases, upon formation of the one or more guanidylated amino acid (e.g., by conjugating to the degradation agent described herein), the one or more amino acids can be degraded (e.g, cleaved) from the polypeptide upon irradiation.

[00214] In some cases, the methods described herein may comprise contacting a terminus of a polypeptide with a degradation agent described herein. In some cases, upon contacting, the polypeptide may comprise a polypeptide (N-terminal amino acid of the polypeptide) coupled to the degradation agent, thereby forming the first modified polypeptide. In some cases, the first modified polypeptide may comprise a photo-cleavable moiety. The method may further comprise subjecting at least a portion of the first modified polypeptide with light. In some cases, the one or more conditions may be exposing at least a portion of the polypeptide to light. In some cases, the light can be directed onto at least a portion of an analyte (e.g., polypeptide). In some cases, the light can be directed to a plurality of analytes. In some cases, the light can be directed to at least a portion of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, less than all of the analytes may come into contact with one or more light sources. In some cases, the light may be directed to the entirety of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, all of the analytes may come into contact with one or more light sources. In some cases, the light can be directed to at least a portion of a polypeptide that is coupled to a support. In some cases, the light can be directed to an entire solution comprising one or more analytes. In some cases, the light can be directed to at least a portion of a solution comprising one or more analytes. If a plurality of analytes in solution, less than all of the analytes may come into contact with one or more light sources. [00215] The method may further comprise subjecting at least a portion of the first modified polypeptide with light having a wavelength from 200 nm to 750 nm. In some cases, the photo- cleavable moiety of the first modified polypeptide may be cleaved upon subjecting to the light having a wavelength from 200 nm to 750 nm. In some cases, the degradation agent described herein may degrade (e.g., cleave) one or more amino acids (of the polypeptide) that are conjugated to the degradation agent when subjected to a light source having a wavelength from about 200 nm to about 750 nm. In some cases, the degradation agent described herein may degrade (e.g., cleave) one or more amino acids (of the polypeptide) that are conjugated to the degradation agent when subjected to a light source having a wavelength about 200 nm to about 250 nm, from about 200 nm to about 300 nm, from about 200 nm to about 350 nm, from about 200 nm to about 400 nm, from about 200 nm to about 450 nm, from about 200 nm to about 500 nm, from about 200 nm to about 550 nm, from about 200 nm to about 600 nm, from about 200 nm to about 650 nm, from about 200 nm to about 700 nm, from about 200 nm to about 750 nm, from about 250 nm to about 300 nm, from about 250 nm to about 350 nm, from about 250 nm to about 400 nm, from about 250 nm to about 450 nm, from about 250 nm to about 500 nm, from about 250 nm to about 550 nm, from about 250 nm to about 600 nm, from about 250 nm to about 650 nm, from about 250 nm to about 700 nm, from about 250 nm to about 750 nm, from about 300 nm to about 350 nm, from about 300 nm to about 400 nm, from about 300 nm to about 450 nm, from about 300 nm to about 500 nm, from about 300 nm to about 550 nm, from about 300 nm to about 600 nm, from about 300 nm to about 650 nm, from about 300 nm to about 700 nm, from about 300 nm to about 750 nm, from about 350 nm to about 400 nm, from about 350 nm to about 450 nm, from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 350 nm to about 650 nm, from about 350 nm to about 700 nm, from about 350 nm to about 750 nm, from about 400 nm to about 450 nm, from about 400 nm to about 500 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 400 nm to about 700 nm, from about 400 nm to about 750 nm, from about 450 nm to about 500 nm, from about 450 nm to about 550 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 450 nm to about 750 nm, from about 500 nm to about 550 nm, from about 500 nm to about 600 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 600 nm, from about 550 nm to about 650 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 600 nm to about 650 nm, from about 600 nm to about 700 nm, from about 600 nm to about 750 nm, from about 650 nm to about 700 nm, from about 650 nm to about 750 nm, or about 700 nm to about 750 nm. In some cases, the degradation agent described herein may degrade (e.g., cleave) one or more amino acids (of the polypeptide) that are conjugated to the degradation agent when subjected to a light source having a wavelength at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, or more. In some cases, the degradation agent described herein may degrade (e.g., cleave) one or more amino acids (of the polypeptide) that are conjugated to the degradation agent when subjected to a light source having a wavelength at most about 250 nm, at most about 300 nm, at most about 350 nm, at most about 400 nm, at most about 450 nm, at most about 500 nm, at most about 550 nm, at most about 600 nm, at most about 650 nm, at most about 700 nm, at most about 750 nm or less.

[00216] In some cases, the polypeptide coupled to a degradation agent may be exposed to a light source (e.g., LED) having a wavelength of from 200 nm to 700 nm. In some cases, the light source has a power output of from about 1 W to about 50 W. In some cases, the light source has a power output of about 1 W to about 5 W, from about 1 W to about 10 W, from about 1 W to about 12 W, from about 1 W to about 20 W, from about 1 W to about 25 W, from about 1 W to about 30 W, from about 1 W to about 35 W, from about 1 W to about 40 W, from about 1 W to about 45 W, from about 1 W to about 50 W, from about 5 W to about 10 W, from about 5 W to about 12 W, from about 5 W to about 20 W, from about 5 W to about 25 W, from about 5 W to about 30 W, from about 5 W to about 35 W, from about 5 W to about 40 W, from about 5 W to about 45 W, from about 5 W to about 50 W, from about 10 W to about 12 W, from about 10 W to about 20 W, from about 10 W to about 25 W, from about 10 W to about 30 W, from about 10 W to about 35 W, from about 10 W to about 40 W, from about 10 W to about 45 W, from about 10 W to about 50 W, from about 12 W to about 20 W, from about 12 W to about 25 W, from about 12 W to about 30 W, from about 12 W to about 35 W, from about 12 W to about 40 W, from about 12 W to about 45 W, from about 12 W to about 50 W, from about 20 W to about 25 W, from about 20 W to about 30 W, from about 20 W to about 35 W, from about 20 W to about 40 W, from about 20 W to about 45 W, from about 20 W to about 50 W, from about 25 W to about 30 W, from about 25 W to about 35 W, from about 25 W to about 40 W, from about 25 W to about 45 W, from about 25 W to about 50 W, from about 30 W to about 35 W, from about 30 W to about 40 W, from about 30 W to about 45 W, from about 30 W to about 50 W, from about 35 W to about 40 W, from about 35 W to about 45 W, from about 35 W to about 50 W, from about 40 W to about 45 W, from about 40 W to about 50 W, or about 45 W to about 50 W. In

-n- some cases, the light source has a power output of at least about 1 W, at least about 5 W, at least about 10 W, at least about 12 W, at least about 20 W, at least about 25 W, at least about 30 W, at least about 35 W, at least about 40 W, at least about 45 W, or more. In some cases, the light source has a power output of at most about 5 W, at most about 10 W, at most about 12 W, at most about 20 W, at most about 25 W, at most about 30 W, at most about 35 W, at most about 40 W, at most about 45 W, at most about 50 W, or less.

[00217] In some cases, the light source may be positioned at a distance of about from 0.05 cm to about 10 cm. In some cases, the light source may be positioned at a distance of about 0.05 cm to about 0.1 cm, from about 0.05 cm to about 0.2 cm, from about 0.05 cm to about 0.4 cm, from about 0.05 cm to about 0.6 cm, from about 0.05 cm to about 0.8 cm, from about 0.05 cm to about 1 cm, from about 0.05 cm to about 2 cm, from about 0.05 cm to about 4 cm, from about 0.05 cm to about 6 cm, from about 0.05 cm to about 8 cm, from about 0.05 cm to about 10 cm, from about 0.1 cm to about 0.2 cm, from about 0.1 cm to about 0.4 cm, from about 0.1 cm to about 0.6 cm, from about 0.1 cm to about 0.8 cm, from about 0.1 cm to about 1 cm, from about 0.1 cm to about 2 cm, from about 0.1 cm to about 4 cm, from about 0.1 cm to about 6 cm, from about 0.1 cm to about 8 cm, from about 0.1 cm to about 10 cm, from about 0.2 cm to about 0.4 cm, from about 0.2 cm to about 0.6 cm, from about 0.2 cm to about 0.8 cm, from about 0.2 cm to about 1 cm, from about 0.2 cm to about 2 cm, from about 0.2 cm to about 4 cm, from about 0.2 cm to about 6 cm, from about 0.2 cm to about 8 cm, from about 0.2 cm to about 10 cm, from about 0.4 cm to about 0.6 cm, from about 0.4 cm to about 0.8 cm, from about 0.4 cm to about 1 cm, from about 0.4 cm to about 2 cm, from about 0.4 cm to about 4 cm, from about 0.4 cm to about 6 cm, from about 0.4 cm to about 8 cm, from about 0.4 cm to about 10 cm, from about 0.6 cm to about 0.8 cm, from about 0.6 cm to about 1 cm, from about 0.6 cm to about 2 cm, from about 0.6 cm to about 4 cm, from about 0.6 cm to about 6 cm, from about 0.6 cm to about 8 cm, from about 0.6 cm to about 10 cm, from about 0.8 cm to about 1 cm, from about 0.8 cm to about 2 cm, from about 0.8 cm to about 4 cm, from about 0.8 cm to about 6 cm, from about 0.8 cm to about 8 cm, from about 0.8 cm to about 10 cm, from about 1 cm to about 2 cm, from about 1 cm to about 4 cm, from about 1 cm to about 6 cm, from about 1 cm to about 8 cm, from about 1 cm to about 10 cm, from about 2 cm to about 4 cm, from about 2 cm to about 6 cm, from about 2 cm to about 8 cm, from about 2 cm to about 10 cm, from about 4 cm to about 6 cm, from about 4 cm to about 8 cm, from about 4 cm to about 10 cm, from about 6 cm to about 8 cm, from about 6 cm to about 10 cm, or about 8 cm to about 10 cm. In some cases, the light source may be positioned at a distance of at least about 0.05 cm, at least about 0.1 cm, at least about 0.2 cm, at least about 0.4 cm, at least about 0.6 cm, at least about 0.8 cm, at least about 1 cm, at least about 2 cm, at least about 4 cm, at least about 6 cm, at least about 8 cm, or more. In some cases, the light source may be positioned at a distance of at most about 0.1 cm, at most about 0.2 cm, at most about 0.4 cm, at most about 0.6 cm, at most about 0.8 cm, at most about 1 cm, at most about 2 cm, at most about 4 cm, at most about 6 cm, at most about 8 cm, at most about 10 cm, less.

[00218] In some cases, upon subjecting the first modified polypeptide (e.g., the polypeptide coupled to a degradation agent) with a light having a wavelength of from 200 nm to 750 nm may be sufficient to generate a second modified polypeptide and a fragment comprising residue of the terminus of the polypeptide. In some cases, the second modified polypeptide may comprise one or more fewer amino acids than the first polypeptide. In some cases, one or more light sources may be used. In some cases, the one or more light source may be used with one or more photomasks, waveguides, spatial light modulators, or digital micromirror devices. In some cases, the one or more light sources may be configured to emit electromagnetic radiation in the ultraviolet, visible, or infrared spectrum. In some cases, the one or more light sources may be employed to initiate, modulate, or facilitate a photoreaction, photodegradation, fluorescence excitation, or other optically responsive processes described herein. In some cases, the light source may include, without limitation: light-emitting diodes (LEDs), laser diodes, solid-state lasers, gas lasers, halogen lamps, incandescent bulbs, and/or sunlight.

[00219] In some cases, the degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety) may be stable at a temperature of from about -40 °C to about 0 °C in the absences of light. In some cases, the degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety) may be stable from about 0 °C to about -5 °C, from about 0 °C to about -10 °C, from about 0 °C to about -15 °C, from about 0 °C to about -20 °C, from about 0 °C to about -25 °C, from about 0 °C to about -30 °C, from about 0 °C to about -35 °C, from about 0 °C to about -40 °C, from about -5 °C to about -10 °C, from about -5 °C to about -15 °C, from about -5 °C to about -20 °C, from about -5 °C to about -25 °C, from about -5 °C to about -30 °C, from about -5 °C to about -35 °C, from about -5 °C to about - 40 °C, from about -10 °C to about -15 °C, from about -10 °C to about -20 °C, from about -10 °C to about -25 °C, from about -10 °C to about -30 °C, from about -10 °C to about -35 °C, from about -10 °C to about -40 °C, from about -15 °C to about -20 °C, from about -15 °C to about -25 °C, from about -15 °C to about -30 °C, from about -15 °C to about -35 °C, from about -15 °C to about -40 °C, from about -20 °C to about -25 °C, from about -20 °C to about -30 °C, from about -20 °C to about -35 °C, from about -20 °C to about -40 °C, from about -25 °C to about -30 °C, from about -25 °C to about -35 °C, from about -25 °C to about -40 °C, from about -30 °C to about -35 °C, from about -30 °C to about -40 °C, or from about -35 °C to about -40 °C in the absences of light. In some cases, the degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety) may be stable at a temperature of at least about 0 °C, at least about -5 °C, at least about -10 °C, at least about -15 °C, at least about -20 °C, at least about -25 °C, at least about -30 °C, at least about -35 °C, or more in the absences of light. In some cases, the degradation agent described herein (e.g., a degradation agent comprising a photo-cleavable moiety) may be stable at a temperature of at most about -5 °C, at most about -10 °C, at most about -15 °C, at most about -20 °C, at most about -25 °C, at most about -30 °C, at most about -35 °C, at most about -40 °C, or less in the absences of light.

[00220] In some cases, organic solutions comprising the degradation agent described herein may be stable at -20 °C for from about 0.5 months to about 6 months. In some cases, organic solutions comprising the degradation agent described herein may be stable at -20 °C for about 0.5 months to about 1 month, from about 0.5 months to about 1.5 months, from about 0.5 months to about 2 months, from about 0.5 months to about 2.5 months, from about 0.5 months to about 3 months, from about 0.5 months to about 3.5 months, from about 0.5 months to about 4 months, from about 0.5 months to about 4.5 months, from about 0.5 months to about 5 months, from about 0.5 months to about 5.5 months, from about 0.5 months to about 6 months, from about 1 month to about 1.5 months, from about 1 month to about 2 months, from about 1 month to about 2.5 months, from about 1 month to about 3 months, from about 1 month to about 3.5 months, from about 1 month to about 4 months, from about 1 month to about 4.5 months, from about 1 month to about 5 months, from about 1 month to about 5.5 months, from about 1 month to about 6 months, from about 1.5 months to about 2 months, from about 1.5 months to about 2.5 months, from about 1.5 months to about 3 months, from about 1.5 months to about 3.5 months, from about 1.5 months to about 4 months, from about 1.5 months to about 4.5 months, from about 1.5 months to about 5 months, from about 1.5 months to about 5.5 months, from about 1.5 months to about 6 months, from about 2 months to about 2.5 months, from about 2 months to about 3 months, from about 2 months to about 3.5 months, from about 2 months to about 4 months, from about 2 months to about 4.5 months, from about 2 months to about 5 months, from about 2 months to about 5.5 months, from about 2 months to about 6 months, from about 2.5 months to about 3 months, from about 2.5 months to about 3.5 months, from about 2.5 months to about 4 months, from about 2.5 months to about 4.5 months, from about 2.5 months to about 5 months, from about 2.5 months to about 5.5 months, from about 2.5 months to about 6 months, from about 3 months to about 3.5 months, from about 3 months to about 4 months, from about 3 months to about 4.5 months, from about 3 months to about 5 months, from about 3 months to about 5.5 months, from about 3 months to about 6 months, from about 3.5 months to about 4 months, from about 3.5 months to about 4.5 months, from about 3.5 months to about 5 months, from about 3.5 months to about 5.5 months, from about 3.5 months to about 6 months, from about 4 months to about 4.5 months, from about 4 months to about 5 months, from about 4 months to about 5.5 months, from about 4 months to about 6 months, from about 4.5 months to about 5 months, from about 4.5 months to about 5.5 months, from about 4.5 months to about 6 months, from about 5 months to about 5.5 months, from about 5 months to about 6 months, or about 5.5 months to about 6 months. In some cases, organic solutions comprising the degradation agent described herein may be stable at -20 °C for at least about 0.5 months, at least about 1 month, at least about 1.5 months, at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, or more. In some cases, organic solutions comprising the degradation agent described herein may be stable at -20 °C for at most about 1 month, at most about 1.5 months, at most about 2 months, at most about 2.5 months, at most about 3 months, at most about 3.5 months, at most about 4 months, at most about 4.5 months, at most about 5 months, at most about 5.5 months, at most about 6 months, or less. [00221] In some cases, aqueous solutions comprising the degradation agent described herein may be stable at -20 °C for from about 0.2 months to about 5 months. In some cases, aqueous solutions comprising the degradation agent described herein may be stable at -20 °C for about 0.2 months to about 0.4 months, from about 0.2 months to about 0.6 months, from about 0.2 months to about 0.8 months, from about 0.2 months to about 1 month, from about 0.2 months to about 1.5 months, from about 0.2 months to about 2 months, from about 0.2 months to about 2.5 months, from about 0.2 months to about 3 months, from about 0.2 months to about 3.5 months, from about 0.2 months to about 4 months, from about 0.2 months to about 5 months, from about 0.4 months to about 0.6 months, from about 0.4 months to about 0.8 months, from about 0.4 months to about 1 month, from about 0.4 months to about 1.5 months, from about 0.4 months to about 2 months, from about 0.4 months to about 2.5 months, from about 0.4 months to about 3 months, from about 0.4 months to about 3.5 months, from about 0.4 months to about 4 months, from about 0.4 months to about 5 months, from about 0.6 months to about 0.8 months, from about 0.6 months to about 1 month, from about 0.6 months to about 1.5 months, from about 0.6 months to about 2 months, from about 0.6 months to about 2.5 months, from about 0.6 months to about 3 months, from about 0.6 months to about 3.5 months, from about 0.6 months to about 4 months, from about 0.6 months to about 5 months, from about 0.8 months to about 1 month, from about 0.8 months to about 1.5 months, from about 0.8 months to about 2 months, from about 0.8 months to about 2.5 months, from about 0.8 months to about 3 months, from about 0.8 months to about 3.5 months, from about 0.8 months to about 4 months, from about 0.8 months to about 5 months, from about 1 month to about 1.5 months, from about 1 month to about 2 months, from about 1 month to about 2.5 months, from about 1 month to about 3 months, from about 1 month to about 3.5 months, from about 1 month to about 4 months, from about 1 month to about 5 months, from about 1.5 months to about 2 months, from about 1.5 months to about 2.5 months, from about 1.5 months to about 3 months, from about 1.5 months to about 3.5 months, from about 1.5 months to about 4 months, from about 1.5 months to about 5 months, from about 2 months to about 2.5 months, from about 2 months to about 3 months, from about 2 months to about 3.5 months, from about 2 months to about 4 months, from about 2 months to about 5 months, from about 2.5 months to about 3 months, from about 2.5 months to about 3.5 months, from about 2.5 months to about 4 months, from about 2.5 months to about 5 months, from about 3 months to about 3.5 months, from about 3 months to about 4 months, from about 3 months to about 5 months, from about 3.5 months to about 4 months, from about 3.5 months to about 5 months, or about 4 months to about 5 months. In some cases, aqueous solutions comprising the degradation agent described herein may be stable at -20 °C for at least about 0.2 months, at least about 0.4 months, at least about 0.6 months, at least about 0.8 months, at least about 1 month, at least about 1.5 months, at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, or more. In some cases, aqueous solutions comprising the degradation agent described herein may be stable at -20 °C for at most about 0.4 months, at most about 0.6 months, at most about 0.8 months, at most about 1 month, at most about 1.5 months, at most about 2 months, at most about 2.5 months, at most about 3 months, at most about 3.5 months, at most about 4 months, about 5 months or less. [00222] In some cases, the reaction time of the degradation process (e.g., from conjugating the degradation agent described herein to the degradation of at least one amino acid of the polypeptide) can be completed in about 5 minutes to about 180 minutes.

[00223] The degradation agent described herein may reduce the time required to degrade (e.g., cleave) one or more amino acids of the polypeptide compared to that of other degradation mechanisms (e.g., Edman degradation). In some cases, the reaction time of the degradation process (e.g., from conjugating the degradation agent described herein to the degradation of at least one amino acid of the polypeptide) can be from about 5 minutes to about 15 minutes, from about 5 minutes to about 30 minutes, from about 5 minutes to about 40 minutes, from about 5 minutes to about 50 minutes, from about 5 minutes to about 60 minutes, from about 5 minutes to about 70 minutes, from about 5 minutes to about 80 minutes, from about 5 minutes to about 90 minutes, from about 5 minutes to about 100 minutes, from about 5 minutes to about 120 minutes, from about 5 minutes to about 180 minutes, from about 15 minutes to about 30 minutes, from about 15 minutes to about 40 minutes, from about 15 minutes to about 50 minutes, from about 15 minutes to about 60 minutes, from about 15 minutes to about 70 minutes, from about 15 minutes to about 80 minutes, from about 15 minutes to about 90 minutes, from about 15 minutes to about 100 minutes, from about 15 minutes to about 120 minutes, from about 15 minutes to about 180 minutes, from about 30 minutes to about 40 minutes, from about 30 minutes to about 50 minutes, from about 30 minutes to about 60 minutes, from about 30 minutes to about 70 minutes, from about 30 minutes to about 80 minutes, from about 30 minutes to about 90 minutes, from about 30 minutes to about 100 minutes, from about 30 minutes to about 120 minutes, from about 30 minutes to about 180 minutes, from about 40 minutes to about 50 minutes, from about 40 minutes to about 60 minutes, from about 40 minutes to about 70 minutes, from about 40 minutes to about 80 minutes, from about 40 minutes to about 90 minutes, from about 40 minutes to about 100 minutes, from about 40 minutes to about 120 minutes, from about 40 minutes to about 180 minutes, from about 50 minutes to about 60 minutes, from about 50 minutes to about 70 minutes, from about 50 minutes to about 80 minutes, from about 50 minutes to about 90 minutes, from about 50 minutes to about 100 minutes, from about 50 minutes to about 120 minutes, from about 50 minutes to about 180 minutes, from about 60 minutes to about 70 minutes, from about 60 minutes to about 80 minutes, from about 60 minutes to about 90 minutes, from about 60 minutes to about 100 minutes, from about 60 minutes to about 120 minutes, from about 60 minutes to about 180 minutes, from about 70 minutes to about 80 minutes, from about 70 minutes to about 90 minutes, from about 70 minutes to about 100 minutes, from about 70 minutes to about 120 minutes, from about 70 minutes to about 180 minutes, from about 80 minutes to about 90 minutes, from about 80 minutes to about 100 minutes, from about 80 minutes to about 120 minutes, from about 80 minutes to about 180 minutes, from about 90 minutes to about 100 minutes, from about 90 minutes to about 120 minutes, from about 90 minutes to about 180 minutes, from about 100 minutes to about 120 minutes, from about 100 minutes to about 180 minutes, or from about 120 minutes to about 180 minutes. In some cases, the reaction time of the degradation process (e.g., from conjugating the degradation agent described herein to the degradation of at least one amino acid of the polypeptide) can be from about 5 minutes, about 15 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 120 minutes, or about 180 minutes. In some cases, the reaction time of the degradation process (e.g., from conjugating the degradation agent described herein to the degradation of at least one amino acid of the polypeptide) can be at least about 5 minutes, at least about 15 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 120 minutes, or more. In some cases, the reaction time of the degradation process (e.g., from conjugating the degradation agent described herein to the degradation of at least one amino acid of the polypeptide) can be at most about 1 minute, at most about 2 minutes, at most about 3 minutes, at most about 4 minutes, at most about 5 minutes, at most about 6 minutes, at most about 7 minutes, at most about 8 minutes, at most about 9 minutes, at most about 10 minutes, at most about 11 minutes, at most about 12 minutes, at most about 13 minutes, at most about 14 minutes, at most about 15 minutes, at most about 30 minutes, at most about 40 minutes, at most about 50 minutes, at most about 60 minutes, at most about 70 minutes, at most about 80 minutes, at most about 90 minutes, at most about 100 minutes, at most about 120 minutes, at most about 180 minutes or less.

[00224] In some cases, the terminal amino acid of the polypeptide may be cleaved. Cleaving may expose the terminal amino group of an adj acent (penultimate) amino acid on the polypeptide, whereby the adjacent amino acid can be available for reaction with a new probe. Optionally, the polypeptide may be sequentially cleaved until the last amino acid in the polypeptide.

Surface

[00225] In some cases, one or more analytes (e.g., a polypeptide) may be in a solution. For example, in some cases, one or more analytes may be in a buffer. For example, in some cases, one or more analytes may be in an acidic, a basic, or a neutral buffer. In some cases, the methods disclosed herein may comprise coupling one or more analytes (e.g., polypeptide) to one or more supports or surfaces. In some cases, a terminus (e.g., C-terminus or N-terminus) of the polypeptide can be coupled to the surface or support.

[00226] As used herein, the term a “surface” or “support” may refer to an entity to which a substance (e.g., molecular construct) can be coupled, immobilized, or adsorbed. The solid may be a solid or semi-solid (e.g., gel) support. As a non- limiting example, a surface or support may be a bead, a polymer matrix, a membrane, an array, a microscopic slide, a glass surface, a plastic surface, a transparent surface, hydrogel, a metallic surface, a metal surface, a magnetic surface, a well, a multi-well plate, a planar surface, a nanoparticle, a microparticle, a lantern, a nanomaterial, a resin, a functionalized surface, or any combination thereof. The support may be planar. As an alternative, the support may be non-planar, such as including one or more wells. A bead can be, for example, a marble, a polymer bead (e.g., a polysaccharide bead, a cellulose bead, a synthetic polymer bead, a natural polymer bead), a silica bead, a functionalized bead, an activated bead, a barcoded bead, a labeled bead, a PCA bead, a magnetic bead, or any combination thereof. A bead may be functionalized with a functional motif. Some nonlimiting examples of functional motifs include a capture reagent (e.g., pyridinecarboxyaldehyde (PCA)), a biotin, a streptavidin, a strep-tag II, a linker, or a functional group that can react with a molecule (e.g., an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, or an aldehyde dithiolane. In some cases, one or more analytes may be coupled to a bead comprising an agarose bead (e.g., Sepharose), a magnetic bead, or a polystyrene microsphere. The functional group may couple specifically to an N-terminus or a C-terminus of a peptide. The functional group may couple specifically to an amino acid side chain. The functional group may couple to a side chain of an amino acid (e.g., the acid of a glutamate or aspartate, the thiol of a cysteine, the amine of a lysine, or the amide of a glutamine, or asparagine). The functional group may couple specifically to a reactive group on a particular species, such as a label. In some examples of functionalized beads, the functional motif can be reversibly coupled and cleaved. A functional motif can also irreversibly couple to a molecule. One such type of substrate may be a lantern, which may comprise a solid support comprising peptide capture agents, and a rod for positioning the solid support within a sample. A lantern rod may be manipulatable by a user (e.g., the user may hold the lantern rod) or an instrument. A lantern rod may be configured to connect to a member proximal to a sample volume. For example, a lantern rod may be configured to couple to a clip above a well of a well plate. A lantern solid support may comprise a reactive group of the present disclosure, such as a reactive group selective for cysteine or a peptide C-terminus. A lantern may be dried or frozen with peptides coupled to its solid support, which may stabilize the peptides coupled thereto. Unbound polypeptides may be washed from a lantern solid support. In other cases, one or more analytes may be coupled to a glass comprising a silanized glass, a silica gel, a glass slide, or a microarray surface. In some cases, one or more analytes may be coupled to a plastic or a polymer comprising polystyrene, polyethylene glycol (PEG), PMMA, or PDMS. In some cases, one or more analytes may be coupled to a membrane comprising nitrocellulose, PVDF, a nylon membrane. In some cases, one or more analytes may be coupled to a hydrogel comprising PEG-based hydrogels, a polyacrylamide, or alginate. In some cases, one or more analytes may be coupled to a metal surface comprising gold, titanium, or stainless steel. In some cases, one or more analytes may be coupled to a nanomaterial comprising a carbon nanotube, graphene oxide, a quantum dot, or a silica nanoparticle. In other cases, one or more analytes may be coupled to one or more resins comprising cross-linked polystyrene, tentagel, or Wang/PEG resin.

[00227] In some cases, the polypeptide may be coupled to a solid support or surface through covalent or non-covalent interactions. The surface or support may be functionalized (e.g., functional group) with a reagent. For example, the functional group may couple specifically to an N-terminus or a C-terminus of a polypeptide. The functional group may couple specifically to an amino acid side chain. The functional group may couple to a side chain of an amino acid (e.g., the acid of a glutamate or aspartate, the thiol of a cysteine, the amine of a lysine, or the amide of a glutamine, or asparagine). The functional group may couple specifically to a reactive group on a particular species, such as a probe. For example, the functional motif can be reversibly coupled and cleaved. A functional group can also irreversibly couple to a molecule. Coupling of the polypeptide to the surface may be achieved via a variety of chemistries, including, but not limited to, carbodiimide-mediated amide bond formation, Michael addition between thiols and malemides, nucleophilic attack on epoxy groups, or biorthogonal click chemistry reaction such as azide-alkyne cycloaddition.

[00228] In some cases, the methods, compositions, systems, and/or kits described herein may comprise a plurality of polypeptides coupled to one or more surfaces or supports. For example, in some cases, the methods, compositions, systems, and/or kits described herein may comprise a first polypeptide and a second polypeptide. In some cases, the first polypeptide may be coupled to a first location on a first support. In some cases, the second polypeptide may be coupled to a second location on a second support. In some cases, the methods may comprise selectively subjecting the at least the portion of the polypeptide to the light at a first time and subjecting at least a portion of the second polypeptide to another light at a second time. In some cases, a distance between the first location and the additional location can be at least about 50 nm, at least about 80 nm, at least about 100 nm, at least about 120 nm, at least about 150 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 1 mm, at least about 5 mm, at least about 10 mm, at least about 20 mm, at least about 30 mm, at least about 40 mm, at least about 50 mm, at least about 100 mm, at least about 500 mm, at least about 1 cm, at least about 5 cm, at least about 10 cm or more. In some cases, a distance between the first location and the additional location can be at most about 50 nm, at most about 80 nm, at most about 100 nm, at most about 120 nm, at most about 150 nm, at most about 200 nm, at most about 300 nm, at most about 400 nm, at most about 500 nm, at most about 1 mm, at most about 5 mm, at most about 10 mm, at most about 20 mm, at most about 30 mm, at most about 40 mm, at most about 50 mm, at most about 100 mm, at most about 500 mm, at most about 1 cm, at most about 5 cm, at most about 10 cm or more. The first location and the second location (that are coupled to the first polypeptide and the second polypeptide, respectively) may be sufficiently positions such that one or more conditions (e.g., one or more conditions sufficient to generate one or more cleaved polypeptides) can be selectively applied to the first location but not to the second location. The distance between the first location and the second location may provide sufficient distance such that one or more conditions (e.g., light) is selectively applied to the second location but not to the first location. For example, in some cases, the methods may comprise selectively subjecting at least a portion of the first polypeptide to a condition (e.g., light), while not subjecting at least a portion of the second polypeptide to the same condition.

Probes

[00229] Provided herein are methods, systems, compositions, and/or kit for providing one or more analytes (e.g., a polypeptide) comprising (e.g., coupled to) one or more probes. For example, in some cases, a polypeptide may comprise one or more probes coupled to one or more amino acids of the polypeptide. The polypeptide may comprise at least one amino acid having at least one probe coupled (e.g., covalently or non-covalently) thereto.

[00230] In some cases, the probe described herein may include, but are not limited to, small molecules, isotopically labeled compounds, or affinity -based reagents. In some cases, the probe may incorporate a reporter group (e.g., a mass tag or stable isotopes). In some cases, the probe described herein may selectively bind to reactive side chains, such as primary amines (e.g., lysine), thiols (e.g., cysteine), or carboxylic acids (e.g., glutamic and aspartic acids). In some cases, the probe described herein may selectively bind to polar amino acids or non-polar amino acids. In some cases, the probe described herein may selectively bind to negative amino acids, positive amino acids, or neutral amino acids. Table 2 shows a classification of the 20 natural amino acids. In some cases, the probe can be coupled to an unnatural amino acid. For example, the probe can be coupled to a post-translationally modified amino acid, such as citrullinated, methylated, sulfurylated, phosphorylated, succinylated, glycosylated, palmitoylated, prenylated, acylated, amidated, hydroxylated, iodinated, chlorinated, fluorinated, nitrosylated, glutathionylated, malonated, biotinylated, oxidized, reduced, or any combination thereof.

Table 2: Classification of 20 amino acids

[00231] In other cases, the probe described herein may be incorporated at site-specific amino acid (e.g., an internal amino acid, a C-terminus amino acid, or N-terminus amino acid). In some cases, a probe may be coupled to proteins or polypeptides through selective reaction with internal amino acid side chains, the N-terminus, or the C-terminus. Suitable reactive groups for internal amino acid residues may include, but are not limited to, N-hydroxysuccinimide (NHS) esters for reaction with lysine residues and N-terminal primary amines, mal eimide or iodoacetamide groups for selective conjugation to thiol-containing residues such as cysteine, and diazonium salts or sulfonation agents for conjugation to tyrosine residues. In some cases, tryptophan residues may be modified using N-bromosuccinimide (NBS), while histidine residues may be derivatized using reagents such as diethylpyrocarbonate (DEPC). Serine and threonine residues may be oxidized with periodate or modified with tosyl-based reagents to facilitate coupling. In some cases, N-terminal probing of polypeptides or proteins may be achieved through reaction with amine-reactive probes such as NHS esters, aldehyde-containing dyes, isothiocyanates, pyrylium salts, or fluorogenic agents such as fluorescamine. For example, NHS esters such as NHS-BODIPY or NHS-Alexa Fluor dyes may form stable amide bonds with the a-amino group at the N-terminus. Alternatively, aldehyde-functionalized probes may form Schiff base intermediates with the N-terminal amine, which may be stabilized by reduction to form secondary amine linkages. Pyrylium-based probes may be used to achieve selective reaction with the N-terminal a-amine over side-chain s-amines of lysine. In some cases, isothiocyanate-functionalized dyes such as fluorescein isothiocyanate (FITC) may be used to label primary amines via thiourea bond formation. A probe that reacts with C-terminal amino acid may react with the terminal carboxyl group using carbodiimide coupling chemistry, such as the use of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in combination with NHS to form a reactive NHS ester intermediate. This intermediate may be reacted with amine- functionalized probes or dyes to form a stable amide bond at the C-terminus. In other cases, C- terminal labeling may be performed via oxime ligation using aminooxy- or hydrazine- functionalized dyes, optionally after oxidative activation of the terminal carboxyl group. In some cases, azide- or alkyne-modified C-termini may be employed to enable bioorthogonal click chemistry with suitably functionalized probes.

[00232] In some cases, the probe described herein may be coupled to hydroxyl, a carboxylic, an amino, or a thiol group of the amino acid of the polypeptide. In some cases, the probe described herein may selectively bind to specific amino acids. For example, the amino acid specific probe may be a methionine specific probe, an arginine specific probe, a histidine specific probe, a tyrosine specific probe, a carboxylic acid R-group specific probe, a lysine specific probe, a cysteine specific probe, a tryptophan specific probe, or any combination thereof. In other cases, the amino acid specific probe comprises a non-natural amino acid specific probe. In some cases, the non-natural amino acid specific probe may be a phosphoserine specific probe, phosphothreonine specific probe, pyroglutamic acid specific probe, hydroxyproline specific probe, azidolysine specific probe, or dehydroalanine specific probe. [00233] Table 3 provides a non-limiting example of amino acid-specific probes that may be used to selectively react with specific amino acid residues in a polypeptide.

Table 3. Amino acid-specific probes

[00234] In some cases, described herein are probes that can selectively be coupled to specific amino acid (e.g., cysteine) or groups of amino acid types (e.g., carboxylate side chain containing amino acids). Non-limiting examples of cysteine specific probe may include certain iodoacetamides, thiols, benzyl and allyl halides, selenocyanates, mal eimides, and alkynes (e.g., certain alkynoic amides). In some cases, a maleimide may be configured to couple to cysteine and lysine. In some cases, lysine-specific probes may include, for example, certain thiocyanates and isothiocyanates, mal eimides, aldehydes, isatoic anhydrides, and NHS esters. For example, a lysyl butylamine sidechain may be selectively coupled to an NHS ester. In some cases, peptide carboxylates (e.g., glutamate, aspartate, and C-terminal carboxylates) may be coupled to the probe through nucleophilic coupling steps. An example of such a coupling process may include carboxyl conversion to amide conversion via amine- based nucleophilic substitution. In some cases, tyrosine-specific probes may include coupling one or more tyrosine-specific probes to the tyrosine phenol hydroxyl carbon through a two-step labeling process using a bifunctional diazonium reagent. Following diazo-coupling to tyrosine, a second reagent (such as a dithiolane) may optionally be coupled to the diazo label (e.g., to selectively couple a detectable moiety to the labeled tyrosine). Alternatively, the diazonium reagent may comprise a detectable moiety or may lack chemically reactive handles for further coupling. In some cases, histidine imidazole nitrogen can be labeled through a two-step labeling process using an alpha-beta unsaturated carbonyl compound, such as 2-cyclohexenone. The alpha-beta unsaturated carbonyl compound may react with histidine in a nucleophilic addition reaction. The alpha- beta unsaturated carbonyl may comprise a detectable moiety. Following histidine coupling, the alpha-beta unsaturated carbonyl may be further coupled to an additional label, such as a dithiolane. Histidine may alternatively be selectively coupled to an epoxide reagent. In some cases, an arginine guanidinium can be acylated (e.g., labeled with an NHS ester with the aid of Barton’s base). In some cases, methionine may comprise a relatively low nucleophilicity and can often be selectively labeled by a redox based scheme where an oxaziridine group reacts specifically with a methionine thioether without cross-reacting with cysteine. In some cases, a tryptophan indole may couple to a diazopropanoate ester, yielding a tertiary amine derivatized tryptophan, The coupling may be metal-catalyst mediated, for example by a dirhodamine(II) tetraacetate complex, which may enhance the selectivity for tryptophan over other amino acid types. In other cases, phosphorylated amino acids such as phosphoserine, phosphotyrosine, or phosphothreonine can be selectively labeled. Such a labeling method may distinguish between types of phosphorylated amino acids. For example, a phosphoryl beta-elimination followed by a label conjugate addition (e.g., a Michael acceptor reaction) step for selectively labeling of phosphoserine (pSer) and phosphothreonine (pThr) over other phosphorylated amino acids such as phosphotyrosine (pTyr). A subsequent pan-phospho labeling method can be implemented to label pTyr.

[00235] For example, the methods described herein may comprise providing a plurality of polypeptides on a solid support. In some cases, amino acids of an amino acid type of the plurality of immobilized polypeptide may comprise one or more probes. In some cases, the amino acid type may be at least one of lysine, cysteine, histidine, and tyrosine. In some cases, the amino acid type may be any one of twenty natural amino acids. In some cases, the amino acid type may be any one of unnatural amino acids (e.g., post-translationally modified amino acids). In some cases, the method further may comprise contacting N-terminal amino acids of the plurality of immobilized polypeptides with a degradation agent under conditions sufficient to remove the N-terminal amino acids of the plurality of immobilized polypeptides. For example, in some cases, the degradation agent may be a degradation agent described herein. In some cases, the degradation agent may be a degradation agent comprising a photo-cleavable moiety. In other cases, the degradation agent may be an Edman degradation agent. In some cases, the methods described herein may comprise detecting one or more signals or signal change of the probe on amino acids of the amino acid type of the plurality of immobilized polypeptide. In some cases, the methods described herein (e.g., providing the plurality of immobilized polypeptides comprising one or more amino acids coupled to one or more probes, contacting N- terminal amino acids with a degradation agent, and detecting one or more signals or signal change) may be repeated.

[00236] In some cases, the methods described herein may comprise providing a polypeptide. In some cases, the polypeptide may comprise a first probe configured to couple to a first amino acid (or an amino acid type). In some cases, the polypeptide may comprise a second probe configured to couple to a second amino acid (or an amino acid type). In some cases, the polypeptide may be immobilized directly or indirectly to a surface or a support. In some cases, the first probe and the second probe may be configured to generate signals or signal change upon binding to the first amino acid or to the second amino acid. In some cases, the methods described herein may comprise detecting one or more signals or signal change associated with the first probe (e.g., coupled to the first amino acid) or the second probe (e.g., coupled to the second amino acid) from the polypeptide. In some cases, the methods may comprise identifying, using at least one of the signals or the signal change, at least a portion of the sequence of the polypeptide. In some cases, the first amino acid may have greater nucleophilicity than the second amino acid. In some cases, one or more amino acids of the polypeptide (e.g., comprising the probe-amino acid conjugates) may be removed. In some cases, the subjecting the polypeptide to conditions sufficient to remove at least one amino acid from the polypeptide may occur before the detecting one or more signs or signal change associated with the first probe or the second probe.

[00237] In some cases, the optical reporter probe described herein may comprise ClpS proteins or modified ClpS proteins. In some cases, the ClpS proteins may be conjugated to different fluorophores. In some cases, the ClpS proteins (e.g, modified ClpS proteins) may selectively recognize particular amino acids at the N-terminus (e.g ₄ N-terminal lysine, arginine, cysteine, or serine), thereby achieving partial fluorosequencing.

[00238] In some cases, a probe described herein (e.g., a universal probe) may be configured to couple to all amino acids (e.g., any one of natural amino acids or unnatural amino acids). For example, in some cases, a probe described herein may be configured to couple to any amino acid residues at the terminal position of a polypeptide, including the N-terminal and/or C-terminal residue. In some cases, the universal probe (e.g., a modified 8-thioester-BODIPY described herein or any one of molecules described in FIG. 19) described herein may be configured to couple to any one of the amino acids (natural amino acids or unnatural amino acids) positioned at the N-terminus. . In some cases, the universal probe described herein may be configured to couple to any one of the amino acids (natural amino acids or unnatural amino acids) positioned at the C-terminus.

[00239] In some cases, an optical reporter probe described herein can be a boron- dipyrromethene dye (BODIPY). In some cases, the optical reporter probe may be a dipyrromethene-BF2 derivative (e.g. ₄ a modified 8-thioester-BODIPY or any one of molecules described in FIG. 19). In some cases, the probe described herein can interact (e.g., via covalent interactions or non-covalent interactions) with one or more amino acids of the polypeptide to form fluorescent conjugated species. For example, the probe described herein can interact with one or more amino acids. The probe may interact with any of the amino acids at the N-terminus. The probe may interact with any of the amino acids at the C-terminus. The probe may interact with N-terminal histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, or serine in a polypeptide.

[00240] In some cases, a probe described herein can comprise Formula VI, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ can be each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cynao, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ can be a labile or leaving group, and any alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio, can be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5 or 6) independently selected substituents from the group consisting of halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some cases, when R^8S can be methyl then: (a) R¹, R², R³, R⁵, R⁶ and R⁷ are not H; (b) R¹ R², R⁶ and R⁷ are not H, and R³ and R⁵ are not methyl, ethyl, or /2-nitrophenyl; (c) R¹, R³, R⁵ and R⁷ are not methyl, and R² and R⁶ are not H; (d) R¹ and R⁷ are not H, and R², R³, R⁵ and R⁶ are not Cl or Br; (e) R³, R⁵, R⁶ and R⁷ are not H, and R² is not — C(O)H; (f) R¹ is not -nitrophenyl, R², R⁶ and R⁷ are not H, and R³ and R⁵ are not methyl; and (g) R¹, R², R⁵, R⁶ and R⁷ are not H, and R³ is not phenyl, /2-nitrophenyl, p- iodophenyl, p-carboxylicphenyl or /2-methoxyphenyl. In some cases, when R^8S is allyl, ethyl, propyl, butyl, t-butyl, n-dodecyl, phenyl, 2,6- dimethylphenyl, p-methylphenyl, p-methoxyphenyl, p-nitrophenyl or benzyl (-CH2PH) then R¹, R², R³, R⁵, R⁶ and R⁷ are not H. In some cases, when R^8S is Cl then (a) R¹, R², R³, R⁵, R⁶ and R⁷ are not H; (b) R¹, R³, R⁵, R⁶ and R⁷ are not H, and R² is not Cl; (c) R³, R⁵ and R⁷ are not H, and R² and R⁵ are not Cl; (d) R¹, R⁵ and R⁷ are not H, and R², R³ and R⁵ are not Cl; and (e) R², R³, R⁵, R⁶ and R⁷ are not Cl. In some cases, R8 is a labile or leaving group.

[00241] In some cases, R⁸ may be SR, SOR, SO2R. In some cases, R⁷ and R¹ may be hydrogen. In some cases, R², R³, R⁵, and R⁶ may be each independently selected from the group consisting of aryl, alkyl, OR, SR, and NR. For example, In some cases, a probe can be Formula VII. In some cases, a probe may be any one of the molecules described in FIG. 19. Formula VII

[00242] A probe may comprise a detectable moiety. The detectable moiety may be optical reporter probe (e.g., fluorescent, phosphorescent, luminescent, or light absorbing). The detectable moiety may be electrochemically detectable (e.g., a redox active moiety with a characteristic oxidation or reduction potential). The detectable moiety may comprise a mass tag (e.g., for identification with mass spectrometry). A plurality of probes may comprise a plurality of detectable moieties. For example, the methods, systems, compositions, and/or kits described herein may involve a plurality of types of probes, each configured to couple to different amino acids, comprising a different detectable moiety that uniquely identifies the label by its type. [00243] In some cases, the methods, systems, compositions, and/or kit described herein may comprise detecting one or more signals or signal change from the one or more amino acids labeled with the probe described herein to identify a characteristic of at least a portion of the polypeptide at the single molecule level. In some cases, one or more characteristics may comprise identifying sequences of a polypeptide. In some cases, the sequence information (or at least a portion of the sequence information) may be used to infer or determine one or more characteristics of the at least the portion of the polypeptide. Such characteristics may include, but are not limited to, the identity, function, structural features, evolutionary origin, post- translational modification sites, and biochemical properties of the polypeptide. In some cases, one or more characteristics may comprise a number of polypeptides in a sample, types of polypeptides in a sample, an origin of a sample, impurities in a sample, the presence or absence of a polypeptide, or any combination thereof. In other cases, the one or more characteristics of the polypeptide may provide insights into subcellular localization signals, membrane-spanning regions, or signal peptides.

[00244] In some cases, the probe can be an optical reporter probe (e.g., a fluorescent probe). In some cases, an optical reporter probe can be used for fluorescence-based detection and imaging. For example, in some cases, the probe may exhibit different fluorescent spectral properties (e.g., optical signal) when conjugated to different terminal (e.g., C-terminal or N- terminal) amino acids. Such probes may incorporate fluorescent dyes that enable real-time or end-point readouts of polypeptide sequences, localization, interactions, or modification status. [00245] In some cases, the optical signal from the probe-amino acid conjugate may be detected while the conjugate (one or more probes coupled to one or more amino acids) is still part of (e.g., bound) the intact polypeptide (e.g., prior to degradation).

[00246] In other cases, the optical signal may be detected after the polypeptide undergoes degradation step of removing terminal amino acids (e.g., via enzymes, chemicals, or a degradation agent described herein). During such processes, the probe-amino acid conjugate may be cleaved from the polypeptide backbone as a labeled fragment, and the release or positional change of the optical signal may be exhibiting a detectable fluorescence signal. [00247] The methods provided herein may comprise a polypeptide comprising one or more amino acids coupled to one or more probes, wherein the one or more probes are further coupled to making moieties, fluorophores, quenchers, or cleavable linker. In some cases, the detection of the probe-amino acid conjugate may be modulated through the use of environment-sensitive fluorophores, quenchers, or cleavable linkers. For example, a fluorophore-amino acid conjugate may remain non-fluorescent due to proximity to a quencher group until degradation (e.g., proteolytic cleavage) separates the quencher from the fluorophore-amino acid conjugate, resulting in a detectable fluorescence signal.

[00248] In some cases, the probe described herein (e.g., a fluorescent probe) may be configured to exhibit different spectral properties when conjugated to different amino acids. In some cases, the different spectral properties (e.g., signal or signal change) may be used to identify a characteristic of at least a portion of the polypeptide.

[00249] In some cases, the at least one labeled internal amino acid comprises an amino acid having a label covalently attached thereto, which label generates the at least one signal or signal change. In some cases, the at least one labeled internal amino acid comprises an amino acid having a probe coupled thereto, which generates the at least one signal or signal change. In some cases, the at least one signal or signal change can be an optical signal. In some cases, the at least one signal or signal change can be detected with an optical detector having single-molecule sensitivity. In some cases, the at least one signal or signal change comprises a plurality of signals of different intensities. In some cases, the at least one signal or signal change comprises a plurality of signals of different frequencies or frequency ranges.

[00250] In some cases, the methods, compositions, systems, and/or kits described herein may comprise using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide. In some cases, at least one signal or signal change (e.g., of an optical signal) may comprise changes in one or more spectral properties (e.g., fluorescence emission intensity, polarity/anisotropy or lifetime). As used herein, the term “spectral properties” may refer to a detectable change in the emission intensity, polarity/anisotropy or lifetime at a single wavelength or at a plurality of wavelengths of a probe conjugated to a terminal amino acid (e.g., N-terminal or C-terminal) or an internal amino acid relative to one or more different terminal amino acids (e.g., N-terminal or C-terminal) or an internal amino acid. For example, in some cases, spectral properties may include spectral shape or peak intensity and/or polarity.

[00251] In some cases, the detecting the one or more signals or signal change comprises detecting one or more spectral properties for each probe conjugated to a terminal amino acid. In some cases, the methods described herein may include detecting fluorescence of the probe bound to the N-terminal amino acid of the polypeptide. In some cases, the methods described herein may include detecting fluorescence of the probe bound to the C-terminal amino acid of the polypeptide. In some cases, the methods described herein may include detecting fluorescence of the probe bound to the internal amino acid of the polypeptide.

[00252] In some cases, the methods described herein comprise detecting the one or more signals or signal change comprises detecting fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof at a single wavelength or at a plurality of wavelengths. Fluorescence may refer to the ability of certain molecules, such as organic probes, to absorb light at a particular wavelength and, after a brief interval, emit light at a different (longer) wavelength. Fluorescence properties that can be precisely measured include fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof. To detect a particular protein target, fluorophores can be usually covalently coupled to an antibody or a probe. Due to diffraction of the probe emission wave, the smallest features normally resolvable by microscopy can be ~250 nm in the lateral (x-y) plane. Overlapping concurrent emissions from adjacent probes usually obscures smaller features, preventing determination of individual components present in structures like the cell membrane, nucleus or cytoskeleton, or multiprotein complexes. However, super-resolution imaging techniques may provide the precise localization of individual fluorescently labeled protein molecules. Methods like STORM may achieve sub-diffraction resolution by spatially and temporally separating the fluorescence emission of individual fluorophores through reversible, stochastic transitioning of only a small fraction from a dark (off) state to a bright (on) state, such that only one molecule can be detected per diffraction-limited area. Using ultrasensitive digital cameras to detect these transient low intensity signals at high speed, the imaging process can be repeated until all probes present in a field of view are detected sequentially, typically over 10,000+ frames that are each populated with a sparse subset of probe emissions. Individual molecules can be then precisely localized using software to fit centroids over each signal, from which a final super-resolution image can be reconstructed. While compatible with live cell or 3D imaging, single molecule imaging may require highly selective probes (e.g. antibodies), and only limited target multiplexing (simultaneous detection of different proteins) has been achieved.

[00253] In some cases, the detecting the one or more signals or signal change may comprise detecting the one or more signals or signal change at a single wavelength. In other cases, the detecting the one or more signals or signal change may be at a plurality of wavelength. For example, a plurality of wavelengths may include at least two wavelengths that are different (e.g., no overlapping spectra of the wavelength) with one another. For example, in some cases, two wavelengths may be used for detecting two different probes bound to two different amino acids or ammo acid types. In other cases, a plurality of wavelengths may include at least two wavelengths that are different from one another, at least a portion of the spectra of the wavelength overlaps. In some cases, a single wavelength or a plurality of wavelength may be emitted from one or more light sources. The light sources may be of the same or different types and may include, without limitation, lasers, light-emitted diodes (LED), or other optical sources capable of generating electromagnetic radiation at the desired wavelengths.

[00254] In some cases, the one or more signals or signal change may be detected at a single wavelength or at plurality of wavelengths from about 200 nm to about 750 nm. In some cases, the one or more signals or signal change may be detected at a single wavelength or at plurality of wavelengths from about 200 nm to about 250 nm, from about 200 nm to about 300 nm, from about 200 nm to about 350 nm, from about 200 nm to about 400 nm, from about 200 nm to about 450 nm, from about 200 nm to about 500 nm, from about 200 nm to about 550 nm, from about 200 nm to about 600 nm, from about 200 nm to about 650 nm, from about 200 nm to about 700 nm, from about 200 nm to about 750 nm, from about 250 nm to about 300 nm, from about 250 nm to about 350 nm, from about 250 nm to about 400 nm, from about 250 nm to about 450 nm, from about 250 nm to about 500 nm, from about 250 nm to about 550 nm, from about 250 nm to about 600 nm, from about 250 nm to about 650 nm, from about 250 nm to about 700 nm, from about 250 nm to about 750 nm, from about 300 nm to about 350 nm, from about 300 nm to about 400 nm, from about 300 nm to about 450 nm, from about 300 nm to about 500 nm, from about 300 nm to about 550 nm, from about 300 nm to about 600 nm, from about 300 nm to about 650 nm, from about 300 nm to about 700 nm, from about 300 nm to about 750 nm, from about 350 nm to about 400 nm, from about 350 nm to about 450 nm, from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 350 nm to about 650 nm, from about 350 nm to about 700 nm, from about 350 nm to about 750 nm, from about 400 nm to about 450 nm, from about 400 nm to about 500 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 400 nm to about 700 nm, from about 400 nm to about 750 nm, from about 450 nm to about 500 nm, from about 450 nm to about 550 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 450 nm to about 750 nm, from about 500 nm to about 550 nm, from about 500 nm to about 600 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 600 nm, from about 550 nm to about 650 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 600 nm to about 650 nm, from about 600 nm to about 700 nm, from about 600 nm to about 750 nm, from about 650 nm to about 700 nm, from about 650 nm to about 750 nm, or about 700 nm to about 750 nm. In some cases, the one or more signals or signal change may be detected at a single wavelength or at plurality of wavelengths that is at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm or more. In some cases, the one or more signals or signal change may be detected at a single wavelength or at plurality of wavelengths at most about 250 nm, at most about 300 nm, at most about 350 nm, at most about 400 nm, at most about 450 nm, at most about 500 nm, at most about 550 nm, at most about 600 nm, at most about 650 nm, at most about 700 nm, at most about 750 nm or less.

[00255] In some cases, one or more probes that can be bound to the one or more amino acids of the polypeptide may be degraded or cleaved. Any suitable methods may be used to cleave one or more probes from the one or more amino acids of the polypeptide. In some cases, the degradation of the one or more probes may comprise using chemical degradation. Alternatively, the degradation of probe may comprise enzymatic degradation with a protease.

Probe Photodegradation

[00256] In other cases, the probe (e.g., BODIPY-based probes) may be removed from the terminus (e.g., N-terminus or C-terminus) thereby leaving the original amino acid (e.g., a free amino acid prior to binding to the probe) after the detection of one or more signals or signal change associated with the original amino acid. In other cases, the probe may be removed from the terminus (e.g., N-terminus or C-terminus) thereby leaving the original amino acid (e.g., a free amino acid prior to binding to the probe) before the detection of one or more signals or signal change.

[00257] In some cases, the probe (e.g., one or more probes coupled to one or more amino acids) may be cleaved from the amino acid by enzymatic digestion or chemical methods. For example, enzymatic digestion may include proteases or esterase. In some cases, chemical methods may include low pH, mild nucleophiles, or subjecting the probe-amino acid conjugates under reducing conditions. In some cases, the probes described herein can be removed (e.g., cleaved) from the amino acid positioned at the terminus (e.g., N-terminus or C-terminus) and the amino acid residue may be recovered upon photoinduced scission in reaction with molecular oxygen as shown below: Peptide

[00258] As shown in the above reaction, the probe described herein can photodegrade (e.g., photobleached) in the presence of molecular oxygen and can release their substitution at the meso position via a beta scission reaction. In some cases, the probe may undergo photodegradation when irradiated (e.g., irradiated at least a portion of the surface or the support and/or at least a portion of the solution) at from about 200 nm to about 750 nm. In some cases, the probe may undergo photodegradation upon irradiation with light having a wavelength from about 200 nm to about 750 nm. In some cases, the probe may undergo photodegradation upon irradiation with light having a wavelength from about 200 nm to about 250 nm, from about 200 nm to about 300 nm, from about 200 nm to about 350 nm, from about 200 nm to about 400 nm, from about 200 nm to about 450 nm, from about 200 nm to about 500 nm, from about 200 nm to about 550 nm, from about 200 nm to about 600 nm, from about 200 nm to about 650 nm, from about 200 nm to about 700 nm, from about 200 nm to about 750 nm, from about 250 nm to about 300 nm, from about 250 nm to about 350 nm, from about 250 nm to about 400 nm, from about 250 nm to about 450 nm, from about 250 nm to about 500 nm, from about 250 nm to about 550 nm, from about 250 nm to about 600 nm, from about 250 nm to about 650 nm, from about 250 nm to about 700 nm, from about 250 nm to about 750 nm, from about 300 nm to about 350 nm, from about 300 nm to about 400 nm, from about 300 nm to about 450 nm, from about 300 nm to about 500 nm, from about 300 nm to about 550 nm, from about 300 nm to about 600 nm, from about 300 nm to about 650 nm, from about 300 nm to about 700 nm, from about 300 nm to about 750 nm, from about 350 nm to about 400 nm, from about 350 nm to about 450 nm, from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 350 nm to about 650 nm, from about 350 nm to about 700 nm, from about 350 nm to about 750 nm, from about 400 nm to about 450 nm, from about 400 nm to about 500 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 400 nm to about 700 nm, from about 400 nm to about 750 nm, from about 450 nm to about 500 nm, from about 450 nm to about 550 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 450 nm to about 750 nm, from about 500 nm to about 550 nm, from about 500 nm to about 600 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 600 nm, from about 550 nm to about 650 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 600 nm to about 650 nm, from about 600 nm to about 700 nm, from about 600 nm to about 750 nm, from about 650 nm to about 700 nm, from about 650 nm to about 750 nm, or about 700 nm to about 750 nm. In some cases, the probe may undergo photodegradation upon irradiation with light having a wavelength at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, or more. In some cases, the probe may undergo photodegradation upon irradiation with light having a wavelength at most about 250 nm, at most about 300 nm, at most about 350 nm, at most about 400 nm, at most about 450 nm, at most about 500 nm, at most about 550 nm, at most about 600 nm, at most about 650 nm, at most about 700 nm, at most about 750 nm, or less.

[00259] In some cases, the one or more conditions may be exposing at least a portion of the polypeptide to light. In some cases, the light can be directed onto at least a portion of an analyte (e.g., polypeptide). In some cases, the light can be directed to a plurality of analytes. In some cases, the light can be directed to at least a portion of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, less than all of the analytes may come into contact with one or more light sources. In some cases, the light may be directed to the entirety of a support that comprises analytes coupled thereto. For example, when a plurality of analytes is coupled to a support, all of the analytes may come into contact with one or more light sources. In some cases, the light can be directed to at least a portion of a polypeptide that is coupled to a support. In some cases, the light can be directed to an entire solution comprising one or more analytes. In some cases, the light can be directed to at least a portion of a solution comprising one or more analytes. If a plurality of analytes in solution, less than all of the analytes may come into contact with one or more light sources.

[00260] In some cases, the probe may undergo photodegradation (e.g., thereby restoring the terminal amino acid) within at least about 1 second, at least about 2 seconds, at least about 5 seconds, at least about 10 seconds, at least about 15 seconds, at least about 30 seconds, at least about 45 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 0.5 hour, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or more post irradiation. In some cases, the probe may undergo photodegradation (thereby restoring the terminal amino acid) in at most about 1 second, at most about 2 seconds, at most about 5 seconds, at most about 10 seconds, at most about 15 seconds, at most about 30 seconds, at most about 45 seconds, at most about 1 minute, at most about 2 minutes, at most about 3 minutes, at most about 4 minutes, at most about 5 minutes, at most about 10 minutes, at most about 15 minutes, at most about 20 minutes, at most about 0.5 hour, at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, or less post irradiation.

[00261] In some cases, one or more light sources may be used. In some cases, the one or more light source may be used with one or more photomasks, waveguides, spatial light modulators, or digital micromirror devices. In some cases, the one or more light sources may be configured to emit electromagnetic radiation in the ultraviolet, visible, or infrared spectrum. In some cases, the one or more light sources may be employed to initiate, modulate, or facilitate a photoreaction, photodegradation, fluorescence excitation, or other optically responsive processes described herein. In some cases, the light source may include, without limitation: light-emitting diodes (LEDs), laser diodes, solid-state lasers, gas lasers, halogen lamps, incandescent bulbs, and/or sunlight.

Samples

[00262] Provided herein are methods, systems, compositions, and/or kits comprising one or more analytes. The analyte may comprise a protein, polypeptide, or polypeptide. For example, in some cases, one or more analytes may be from a sample (e.g., a biological sample). A biological sample may be derived from a subject (e.g., a patient or a participant in a study), from a tissue sample (e.g., an engineered tissue sample), from a cell culture (e.g., a human cell line or a bacterial colony), from a cell (e.g., a cell isolated during a single cell sorting assay), or a portion thereof (e.g., an organelle from a cell or an exosome from a blood sample). In some cases, the biological sample may comprise biological fluids (e.g., peripheral blood sample). In some cases, one or more analytes of the plurality of analytes comprises a polypeptide. In other cases, the polypeptide may be among a sample comprising a plurality of analytes. A sample may be synthetic, such as a composition of synthetic polypeptides. A sample may comprise a single species or a mixture of species. A biological sample may comprise biomaterial from a single organism, from a colony of genetically near-identical organisms, or from multiple organisms (e.g., enterocytes and microbiota from a human digestive tract). A biological sample may be fractionated (e.g., plasma separated from whole blood), filtered, or depleted (e.g., high abundance proteins such as albumin and ceruloplasmin removed from plasma).

[00263] A sample may comprise all or a subset of the biomolecules from the subject, tissue sample, cell culture, cell, or portion thereof. For example, a sample from a subject may comprise the majority of proteins present in that subject, or may comprise a small subset of the proteins from that subject. A biological sample may comprise a bodily fluid such as cerebral spinal fluid, saliva, urine, tears, blood, plasma, serum, breast aspirate, prostate fluid, seminal fluid, stool, amniotic fluid, intraocular fluid, mucous, or any combination thereof. A biological sample may comprise a tissue culture, for example a tumor sample, or tissue from a kidney, liver, lung, pancreas, stomach, intestine, bladder, ovary, testis, skin, colorectal, breast, brain, esophagus, placenta, or prostate.

[00264] The biological sample may comprise a molecule whose presence or absence may be measured or identified. The biological sample may comprise a macromolecule, such as, for example, a polypeptide or a protein. The macromolecule may be isolated (e.g., separated from other components from which it was sourced) or purified, such that the macromolecule comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of a composition by weight (e.g., by dry weight or including solvent). The biological sample may be complex, and may comprise a plurality of components (e.g., different polypeptides, heterogeneous sample from a CSF of a proteopathy patient). The biological sample may comprise a component of a cell or tissue, a cell or tissue extract, or a fractionated lysate thereof. The biological sample may be substantially purified to contain molecules of a single type (such as polypeptides, nucleic acids, lipids, or small molecules). A biological sample may comprise a plurality of polypeptides configured for a method of the present disclosure (e.g., digestion, C-terminal labeling, or fluorosequencing).

[00265] In some cases, the methods, systems, compositions, and/or kits described herein may comprise isolating, enriching, or purifying a biomolecule, bio macromolecular structure (e.g., an organelle or a ribosome), a cell, or tissue from a biological sample. A method may utilize a biological sample as a source for a biological species of interest. For example, an assay may derive a protein, such as alpha synuclein, a cell, such as a circulating tumor cell (CTC), or a nucleic acid, such as cell-free DNA, from a blood or plasma sample. A method may derive multiple, distinct biological species from a biological sample, such as two separate types of cells. In such cases, the distinct biological species may be separated for different analyses (e.g., CTC lysate and buffycoat proteins may be partitioned and separately analyzed) or pooled for common analysis. A biological species may be homogenized, fragmented, or lysed prior to analysis. In particular instances, a species or plurality of species from among the homogenate, fragmentation products, or lysate may be collected for analysis. For example, a method may comprise collecting circulating tumor cells during a liquid biopsy, optionally isolating individual circulating tumor cells, lysing the circulating tumor cells, isolating polypeptides from the resulting lysate, and analyzing the polypeptides by a fluorosequencing method of the present disclosure. A method may comprise capturing polypeptides from a sample using a C-terminal capture reagent, and analyzing the polypeptides (e.g., by a fluorosequencing method).

[00266] In some cases, the analyte described herein may comprise a polypeptide that is coupled to at least one biomolecule or a functional molecule. For example, in some cases, the polypeptide can be coupled to at least one biomolecule or a functional molecule for detection, targeting, therapy, or structural purposes. In some cases, the polypeptide can be coupled to one or more nucleic acid-based molecules For example, nucleic acid-based molecules can be DNAs, RNAs, DNA and/or RNA barcodes, or aptamers. In some cases, the polypeptide can be coupled to one or more chemical and molecular tags. For example, a chemical and molecular tag may be fluorescent tags (e.g., FITC, rhodamine, Alexa Fluor dyes), biotin, enzyme tags (e.g., horseradish peroxidase), affinity tags (e.g., His-tag or Myc-tag), epitope tags (e.g., recognized by specific antibodies), or click chemistry handles (e.g., azide or alkyne). In other cases, the polypeptide can be coupled to one or more therapeutic or bioactive agents. For example, therapeutic or bioactive agents may be small molecule drugs, toxins, enzymes, hormones, cytokines/growth factors, receptor ligands, cellular compartment specific polynucleotides or polypeptides (e.g., cell-penetrating peptides or nuclear localization signals) or antibodies or antigen binding fragment thereof. In some cases, the polypeptide can be coupled to one or more polymers or carriers. For example, polymers or carriers may be polyethylene glycol (PEG), lipids, nanoparticles, hydrogels or scaffolds. In some cases, the polypeptide can be coupled to one or more radioisotopes, quantum dots, metals, or chelators.

[00267] In some cases, the methods, systems, compositions, and/or kits described herein may comprise nucleic acid analysis, such as sequencing, southern blot, or epigenetic analysis. Nucleic acid analysis may be performed in parallel with a second analytical method, such as a fluorosequencing method of the present disclosure. The nucleic acid and the subject of the second analytical method may be derived from the same subject or the same sample. For example, a method may comprise collecting cell free DNA and a polypeptide from a human plasma sample, sequencing the cell free DNA (e.g., to identify a cancer marker), and performing proteomic analysis on the plasma proteins.

[00268] In some cases, the methods disclosed herein can further comprise one or more sample preparation processes. The sample preparation process can comprise extracting a sample from a subject. For example, the biological sample may be obtained using any clinically or experimentally acceptable techniques, including venipuncture, tissue biopsy, swab collection, or lumbar puncture, depending on the sample type. Following collection, proteins or polypeptides can be extracted from the biological sample. In some cases, the biological sample may be subjected to mechanical, chemical, or enzymatic disruption to lyse cells and release intracellular contents, mechanical disruption methods may include sonication bead beating, or homogenization. Chemical lysis may be achieved using detergents, or chaotropic agents such as sodium dodecyl sulfate (SDS), NP-40, or RIPA buffer. In some cases, enzymatic digestion may be used using proteinase K. After lysis, the sample may be clarified by centrifugation or filtration to isolate the protein. In some cases, the extracted proteins or polypeptides may be denatured to unfold their secondary or tertiary structures. For example, in some cases, the denaturation may be achieved by heat, by chemical agents, or by reducing agents. In some cases, the denatured proteins or polypeptides may be fragmented into smaller peptides or polypeptides. In some cases, enzymatic digestion may be employed, such as treatment with trypsin, chymotrypsin, or other proteases that cleaves at specific amino acids residue. In some cases, chemical fragmentation may be used using cyanogen bromide (CNBr) cleavage. The resulting peptides or polypeptides may then be used directly or subject to further purification. In some cases, the resulting peptides or polypeptides may be coupled to a surface or a support described herein.

[00269] In some cases, the method further comprises a step of treating an immobilized polypeptide (e.g., a support or bead) under conditions such that each terminal amino acid (e.g., N-terminal amino acid or C-terminal amino acid) of each polypeptide can be removed by degradation reaction using reagents of the disclosure; and/or a step of detecting the signal for each polypeptide at the single molecule level. In some cases, the terminal amino acid (e.g., N- terminal or C-terminal) removing step and/or the detecting step can be successively repeated from about 1 time to about 5 times, from about 5 times to about 10 times, from about 10 times to about 20 times, from about 20 times to about 30 times, from about 30 times to about 40 times, from about 40 times to about 50 times, from about 50 times to about 60 times, from about 60 times to about 70 times, from about 70 times to about 80 times, from about 80 times to about 90 times, or from about 90 times to about 100 times. In some cases, the N-terminal amino acid removing step and/or the detecting step can be successively repeated at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times. In some cases, the N-terminal amino acid removing step and/or the detecting step can be successively repeated about 5 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times or more. In some cases, the terminal amino acid (an N-terminal or C-terminal) removing step and/or the detecting step can be successively repeated at most about 5 times, at most about 10 times, at most about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times or less.

Data Processing

[00270] In some cases, the methods, systems, compositions, and/or kits described herein may include comparing the spectral properties of the probe bound to an amino acid (e.g., C-terminal or N-terminal amino acids) of a polypeptide to a plurality of reference spectral properties. In some cases, the spectral properties may include, but are not limited to, fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof. In some cases, each reference spectral property can be representative of the probe conjugated to a different amino acid. In some cases, comparing the spectral properties of the probe to the plurality of reference spectral properties may comprise comparing the spectra of the probe bound to the terminal amino acid to a plurality of reference spectra. In some cases, the reference spectra can be spectra of the probe bound to known terminal amino acids. In some cases, the method may comprise identifying the closest match between the spectra of the probe and the reference spectra, thereby identifying the terminal amino acid of the polypeptide. Various statistical methods known in the art may be used to compare the spectra of the probe and reference spectra in order to identify the closest match and the terminal amino acid of the polypeptide.

[00271] In some cases, suitable methods generate a quantitative measure of similarity or difference between the spectra and the reference spectra. In some cases, the methods described herein further comprises generating a statistical measure or probability score that a spectrum can be indicative of the presence of a particular terminal amino acid residue conjugated to the probe. In some cases, the methods used herein for comparing the spectral properties of a terminal amino acid-probe conjugate and a reference/control conjugate use one or more probabilistic algorithms. For example, a probabilistic algorithm can be trained to identify different terminal amino acids conjugated to HMRG-BOC using the spectral data associating specific spectra with specific terminal amino acids. Additional reference data sets suitable for training probabilistic algorithms can also be generated using other probes that exhibit different spectral properties when conjugated to different terminal amino acids. In some cases, machine learning, genetic algorithms, or principal component analysis (PCA) may be used for comparing spectra and reference spectra.

[00272] Different techniques may be used to detect spectral properties of different molecules at spatially resolved locations. For example, super resolution microscopy may be used to detect one or more spectral properties of a probe conjugated to the terminal amino acid at a particular location within a sample. In some cases, the methods described herein use stochastic optical reconstruction microscopy (STORM). In other cases, the detecting the spectra properties of a probe may include ultrasensitive detection systems that are able to repeatedly detect signals from precisely the same coordinates in a sample, thereby assigning the detected spectral information to a unique polypeptide molecule.

[00273] In some cases, the spectral properties can be detected using an optical detection system. Optical detection systems may include super-resolution fluorescence microscopy, electron multiplying CCD (EMCCD), near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, a charge- coupled device (CCD), dark field microscopy, photoconversion, single and/or multiphoton excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, total internal reflection fluorescence (TIRF) microscopy, single-molecule localization microscopy, and single-molecule spectroscopy. In general, methods may involve detection of laser-activated fluorescence using a microscope equipped with a camera, sometimes referred to as high-efficiency photon detection system. Suitable photon detection systems include, but are not limited to, photodiodes and intensified CCD cameras.

[00274] In some cases, examples of techniques suitable for single molecule detection of the spectral properties of probes include fluorescence correlation spectroscopy, wide-field microscopy, near-field microscopy, confocal laser (scanning) microscopy, fluorescence lifetime imaging microscopy, fluorescence intensity distribution analysis, measuring brightness changes induced by quenching/dequenching of fluorescence, or fluorescence energy transfer.

[00275] In some cases, the method may include comparing the sequence obtained for each polypeptide molecule to a reference protein sequence database. In some cases, small fragments comprising 10-20, or fewer, sequenced amino acid residues, consecutive or with gaps, may be useful for detecting the identity of a polypeptide in a sample.

[00276] In another aspect, disclosed herein are various systems and system components that can be used to determine a characteristic of one or more analytes and/or a sample using the methods, compositions, or kits disclosed herein. In some cases, the system may comprise one or more light sources. The system may comprise one or more detectors. In some cases, the system may comprise one or more optical components (e.g., filters, lenses, mirrors). The one or more filters may be used to isolate wavelengths and/or emitted signal from background noise. The system may also comprise one or more controllers operatively coupled any components disclosed herein. The components can comprise one or more of light source, detector, optical components, or any combination thereof. The one or more controllers may be individually or collectively configured to perform any of the methods disclosed herein.

Kits

[00277] Another aspect of the present disclosure provides kits for analyzing one or more samples comprising one or more polypeptides. In some cases, the disclosure provides kits for identifying at least one characteristic (e.g., sequence) of at least a portion of the polypeptide, using a degradation agent described herein (e.g., a degradation agent comprising a photo- cleavable moiety). In some cases, the kits may comprise one or more probes configured to selectively bind to amino acid residue of one or more polypeptides. In some cases, the kits may comprise one or more probes configured to bind to any N-terminal or C-terminal amino acid residues of one or more polypeptides. The kits may further include one or more degradation agents capable of degrading (e.g., cleaving) at least one amino acid residue from a polypeptide. For example, the kits may include a degradation agent described herein (e.g., a degradation agent comprising a photocleavable moiety), chemical reagents (e.g., phenylisothiocyanate for Edman degradation, hydrazine, or cyanogen bromide), enzymatic or chemical reagents (e.g., amino peptidases, carboxypeptidases, or protease with defined cleaving specificities), and/or combination thereof.

[00278] In some cases, the kit may further include reagents and components for immobilizing the polypeptide to a solid support. The solid support may include, but is not limited to, functionalized glass, polymeric substrates, microbeads, membranes, silicon chips, or microfluidic devices comprising reactive functional groups (e.g., aldehyde, carboxyl, epoxy, NHS-ester, or streptavidin/biotin systems) for covalent or non-covalent attachment of the polypeptide. The kit may include surface-activation agents, cross-linkers, capture tags (e.g., His- tags, FLAG-tags, or biotin), or binding buffers to facilitate and stabilize the immobilization of the polypeptide onto the support under appropriate conditions. In other cases, blocking reagents may also be provided to reduce non-specific binding to the support.

[00279] In some cases, the kits may comprise reagents and components for preparing biological or synthetic samples and for isolating one or more polypeptides from the biological or synthetic samples. This may include reagents for cell lysis, protein extraction, purification, enrichment, or any combination thereof. Suitable lysis buffers may comprise detergents, chaotropic agents (e.g., urea), salts, protease inhibitors, or any combination thereof to preserve polypeptide integrity during extraction.

[00280] The kit may additionally comprise one or more reagents comprising buffers, salts, excipients, surfactants, preservatives, stabilizers, cryoprotectants, or any combination thereof. Suitable components include, but are not limited to, phosphate-buffered saline (PBS), Tris-HCl, NaCl, KC1, Tween-20, BSA, glycerol, mannitol, sucrose, polyethylene glycol (PEG), EDTA, DMSO, trehalose, or any combination thereof. These reagents may be included to maintain proper pH, osmolarity, ionic strength, and protein stability throughout the labeling, degradation, and detection processes. The kit may further include buffers, stabilizers, wash solutions, and other reagents that can perform the labeling, degradation, and detection steps, as well as instructions for use. One or more components of the kit may be formulated for storage at low temperatures (e.g., 4°C, -20°C, or -30°C), and may include reagents that are stable for extended periods under such storage conditions.

[00281] The kit may optionally further include hardware or software components, such as optical detection systems, calibration standards, or analytical software configured to interpret emission signals and compare them to reference values for amino acid identification. Instructions for use may be provided in printed or electronic form, detailing procedures for polypeptide immobilization, probe labeling, amino acid degradation, signal acquisition, sequence determination, or any combination thereof.

[00282] The kit may also include a detection system or instructions for using a detection system suitable for monitoring the emission properties of the fluorescent probes, including but not limited to fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof. In some cases, the kit may include a reference database or calibration standards for correlating measured emission properties to known amino acid identities.

[00283] In another aspect, disclosed herein are various kits and kit components that can be used to determine a characteristic of one or more analytes and/or a sample using the methods, compositions, or systems disclosed herein. The kit can comprise one or more components disclosed herein in relation to any of the various aspects, in any combination. Reagents and other components in the kit can be contained in any suitable container. In some cases, the kit can comprise instructions for use of the kit in accordance with one or more methods disclosed herein. The kit according to any aspects or embodiments disclosed herein can comprise one or more buffers disclosed herein, one or more reagents disclosed herein, one or more probes disclosed herein, one or more degradation agents, one or more enzymes, or any combination thereof.

Computer Systems

[00284] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[00285] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 1 shows a computer system 101 that is programmed or otherwise configured to implement methods or parts of methods disclosed herein, including compiling, analyzing, and displaying data obtained through the present methods. The computer system 101 may regulate various aspects of the present disclosure, such as, for example, controlling excitation (e.g., excitation with light of an appropriate wavelength), controlling one or more light sources, and optical imaging devices. The computer system 101 may be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device may be a mobile electronic device.

[00286] The computer system 101 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 105, which may be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 101 also includes memory or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage and/or electronic display adapters. The memory 110, storage unit 115, interface 120 and peripheral devices 125 are in communication with the CPU 105 through a communication bus (solid lines), such as a motherboard. The storage unit 115 may be a data storage unit (or data repository) for storing data. The computer system 101 may be operatively coupled to a computer network (“network”) 130 with the aid of the communication interface 120. The network 130 may be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 130 in some cases is a telecommunication and/or data network. The network 130 may include one or more computer servers, which may enable distributed computing, such as cloud computing. The network 130, in some cases with the aid of the computer system 101, may implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server. [00287] The CPU 105 may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 110. The instructions may be directed to the CPU 105, which may subsequently program or otherwise configure the CPU 105 to implement methods of the present disclosure. Examples of operations performed by the CPU 105 may include fetch, decode, execute, and writeback.

[00288] The CPU 105 may be part of a circuit, such as an integrated circuit. One or more other components of the system 101 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[00289] The storage unit 115 may store files, such as drivers, libraries and saved programs. The storage unit 115 may store user data, e.g., user preferences and user programs. The computer system 101 in some cases may include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet.

[00290] The computer system 101 may communicate with one or more remote computer systems through the network 130. For instance, the computer system 101 may communicate with a remote computer system of a user (e.g., a fluorimeter or a cell sorting device). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user may access the computer system 101 via the network 130.

[00291] Methods as described herein may be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115. The machine executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor 105. In some cases, the code may be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105. In some situations, the electronic storage unit 115 may be precluded, and machine-executable instructions are stored on memory 110.

[00292] The code may be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or may be compiled during runtime. The code may be supplied in a programming language that may be selected to enable the code to execute in a precompiled or as-compiled fashion.

[00293] Aspects of the systems and methods provided herein, such as the computer system 101, may be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code may be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media may include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and/or the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non- transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution. [00294] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier- wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD- ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00295] The computer system 101 may include or be in communication with an electronic display 135 that comprises a user interface (UI) 140 for providing, for example, orders and options for controlling flow rates in a cell sorting device. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[00296] Methods and systems of the present disclosure may be implemented by way of one or more algorithms. An algorithm may be implemented by way of software upon execution by the central processing unit 105. The algorithm may, for example, determine a correlation using linear and quadratic discriminant analysis (LDA and QDA), Support Vector Machine (SVM), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), Naive Bayes, Random Forest, or any other suitable method.

EXEMPLARY EMBODIMENTS

[00297] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[00298] Embodiment 1. A method, comprising: (a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide, comprising a photo- cleavable moiety; and (b) subjecting at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide and a fragment comprising a residue of the terminus of the polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

[00299] Embodiment 2. The method of embodiment 1, wherein the photo-cleavable moiety is cleavable when subjected to light comprising a wavelength from 200 nm to 750 nm.

[00300] Embodiment 3. The method of embodiment 1 or 2, wherein the condition sufficient to generate the second modified polypeptide comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof.

[00301] Embodiment 4. The method of embodiment 3, wherein the buffer condition comprises an aqueous basic buffer.

[00302] Embodiment 5. The method of any one of embodiments 1-4, wherein the condition sufficient to generate the second modified polypeptide comprises the light source and/or the aqueous basic buffer.

[00303] Embodiment 6. The method of embodiment 5, wherein the light source comprises a wavelength from about 200 nm to about 750 nm.

[00304] Embodiment 7. The method of any one of embodiments 4-6, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0.

[00305] Embodiment 8. The method of any one of embodiments 3-7, wherein the temperature comprises a reaction temperature.

[00306] Embodiment 9. The method of embodiment 8, wherein the condition sufficient to generate the second modified polypeptide comprises the reaction temperature from about 30 degrees to 60 degrees.

[00307] Embodiment 10. A method, comprising: (a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide; and (b) subjecting at least a portion of the first modified polypeptide to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

[00308] Embodiment 11. The method of embodiment 10, wherein (b) comprises generating a fragment comprising a residue of the terminus of the polypeptide.

[00309] Embodiment 12. The method of embodiment 10 or 11, wherein the one or more wavelengths is from about 200 nm to about 500 nm.

[00310] Embodiment 13. The method of any one of embodiments 10-12, wherein (b) comprises subjecting the first modified polypeptide to one or more additional conditions sufficient to generate the second modified polypeptide.

[00311] Embodiment 14. The method of any one of embodiments 10-13, wherein the one or more additional conditions comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof.

[00312] Embodiment 15. The method of embodiment 14, wherein the buffer condition comprises an aqueous basic buffer. [00313] Embodiment 16. The method of embodiment 14 or 15, wherein the one or more additional conditions comprises an aqueous basic buffer.

[00314] Embodiment 17. The method of embodiment 16, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0.

[00315] Embodiment 18. The method of any one of embodiments 14-17, wherein the temperature comprises a reaction temperature.

[00316] Embodiment 19. The method of embodiment 18, wherein the one or more additional conditions comprises a reaction temperature from about 30 degrees to 60 degrees.

[00317] Embodiment 20. The method of any one of embodiments 1-19, wherein the degradation agent comprises a photo-cleavable moiety.

[00318] Embodiment 21. The method of any one of embodiments 1-20, wherein the degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein: LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety. [00319] Embodiment 22. The method of embodiment 21, wherein LG is -SCL'.

[00320] Embodiment 23. The method of embodiment 21 or 22, wherein R¹ is hydrogen.

[00321] Embodiment 24. The method of any one of embodiments 21-23, wherein R² is hydrogen.

[00322] Embodiment 25. The method of any one of embodiments 21-24, wherein the photo- cleavable moiety comprises one or more aromatic groups.

[00323] Embodiment 26. The method of any one of embodiments 21-24, wherein the photo- cleavable moiety comprises a nitro- substituted benzyl group.

[00324] Embodiment 27. The method of any one of embodiments 21-24, wherein the photo- cleavable moiety is

[00325] Embodiment 28. The method of any one of embodiments 1-27, wherein the degradation agent is

[00326] Embodiment 29. The method of any one of embodiments 1-27, further comprising, prior to (a), providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes.

[00327] Embodiment 30. The method of embodiment 29, wherein the polypeptide is coupled to a biomolecule.

[00328] Embodiment 31. The method of embodiment 29 or 30, wherein an amino acid of the one or more amino acids is a terminal amino acid.

[00329] Embodiment 32. The method of embodiment 31, wherein the amino acid is an N- terminal amino acid.

[00330] Embodiment 33. The method of embodiment 29 or 30, wherein an amino acid of one or more amino acids is an internal amino acid.

[00331] Embodiment 34. The method of any one of embodiments 29-33, wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids.

[00332] Embodiment 35. The method of embodiment 34, wherein the probe comprises a dipyrromethene-BF2 derivative.

[00333] Embodiment 36. The method of any one of embodiments 29-33, wherein a probe of the one or more probes is specific to an amino acid type.

[00334] Embodiment 37. The method of embodiment 36, wherein the probes comprise caseinolytic protease adaptor S (ClpS) or a modified ClpS.

[00335] Embodiment 38. The method of any one of embodiments 34-37, wherein the probe is covalently coupled to the polypeptide.

[00336] Embodiment 39. The method of any one of embodiments 34-38, wherein the probe is coupled to hydroxyl, a carboxylic, an amino, a thiol group of the amino acid of the polypeptide or any combination thereof.

[00337] Embodiment 40. The method of any one of embodiments 34-39, wherein the probe exhibits different fluorescent spectral properties when conjugated to different terminal amino acids. [00338] Embodiment 41. The method of any one of embodiments 1-40, further comprising detecting one or more signals or signal change from the one or more probes to identify a characteristic of at least a portion of the polypeptide.

[00339] Embodiment 42. The method of any one of embodiments 1-41, wherein the one or more signals or signal change comprises one or more fluorescent spectral properties.

[00340] Embodiment 43. The method of any one of embodiments 1-42, further comprising using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide.

[00341] Embodiment 44. The method of any one of embodiments 41-43, wherein the detecting the one or more signals or signal change comprises detecting one or more spectral properties of the probe conjugated to the terminal amino acid.

[00342] Embodiment 45. The method of any one of embodiments 41-44, wherein the detecting the one or more signals or signal change comprises stochastic optical reconstruction microscopy (STORM).

[00343] Embodiment 46. The method of any one of embodiments 41-45, wherein the detecting the one or more signals or signal change comprises detecting fluorescence of the probe.

[00344] Embodiment 47. The method of any one of embodiments 41-46, wherein the detecting the one or more signals or signal change comprises detecting fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof.

[00345] Embodiment 48. The method of any one of embodiments 41-47, wherein the detecting the one or more signals or signal change is at a single wavelength.

[00346] Embodiment 49. The method of any one of embodiments 41-48, wherein the detecting the one or more signals or signal change is at a plurality of wavelengths.

[00347] Embodiment 50. The method of any one of embodiments 1-49, wherein the polypeptide is coupled to a surface or support.

[00348] Embodiment 51. The method of embodiment 50, wherein the support comprises a bead, a polymer matrix, a membrane, an array, a microscopic slide, a glass surface, a plastic surface, a transparent surface, hydrogel, a metallic surface, a metal surface, a magnetic surface, a well, a multi-well plate, a planar surface, a nanoparticle, a microparticle, a lantern, a nanomaterial, a resin, a functionalized surface or any combination thereof.

[00349] Embodiment 52. The method of embodiment 50 or 51, wherein the terminus of the polypeptide is coupled to the surface or support. [00350] Embodiment 53. The method of embodiment 52, wherein the terminus is a C- terminus of the polypeptide.

[00351] Embodiment 54. The method of any one of embodiments 1-53, further comprising repeating (a) and (b) one or more times to degrade one or more subsequent terminal amino acids of the polypeptide.

[00352] Embodiment 55. The method of any one of embodiments 1-54, wherein the polypeptide is among a sample comprising a plurality of analytes.

[00353] Embodiment 56. The method of embodiment 55, wherein the sample is a biological sample.

[00354] Embodiment 57. The method of embodiment 56, wherein the biological sample comprises biological fluid, a tissue sample, a cell culture, a cell, or a portion thereof.

[00355] Embodiment 58. The method of any one of embodiments 55-57, wherein an analyte of the plurality of analytes comprises a polypeptide.

[00356] Embodiment 59. The method of any one of embodiments 1-58, further comprising providing one or more additional polypeptides comprising one or more probes coupled to one or more amino acids of the one or more polypeptides.

[00357] Embodiment 60. The method of embodiment 59, further comprising contacting one or more additional polypeptides with one or more additional degradation agents, thereby forming one or more additional modified polypeptides.

[00358] Embodiment 61. The method of embodiment 59 or 60, wherein the one or more additional modified polypeptides comprises one or more additional photo-cleavable moieties. [00359] Embodiment 62. The method of any one of embodiments 59-61, further comprising subjecting the one or more additional modified polypeptides to conditions sufficient to generate one or more cleaved polypeptides.

[00360] Embodiment 63. The method of any one of embodiments 59-62, wherein the degradation agent and the one or more additional degradation agents comprise the same chemical structure.

[00361] Embodiment 64. The method of any one of embodiments 59-63, wherein the degradation agent and the one or more additional degradation agents comprise a different chemical structure.

[00362] Embodiment 65. The method of any one of embodiments 1-64, further comprising identifying a terminal amino acid of the polypeptide by comparing one or more spectral properties generated by the probe to a plurality of reference spectral properties. [00363] Embodiment 66. The method of embodiment 65, wherein the one or more spectral properties is representative of a probe conjugated to a different terminal amino acid.

[00364] Embodiment 67. The method of any one of embodiments 1-66, wherein the polypeptide comprises one or more natural amino acids.

[00365] Embodiment 68. The method of any one of embodiments 1-67, wherein the polypeptide comprises one or more unnatural amino acids.

[00366] Embodiment 69. A method for sample analysis, comprising: (a) providing a sample comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is coupled to a first one or more probes and the second polypeptide is coupled to a second one or more probes; (b) detecting (1) one or more signals or signal change from the first one or more probes and (2) one or more signals or signal change from the second one or more probes; (c) contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent, thereby removing at least one amino acid from the first polypeptide and/or from the second polypeptide, wherein the first degradation agent comprises a first photo-cleavable moiety and/or the second degradation agent comprises a second photo-cleavable moiety; and (d) identifying one or more characteristics of the sample. [00367] Embodiment 70. The method of embodiment 69, repeating (a) and (b) one or more additional times on one or more subsequent amino acids of the first polypeptide and/or the second polypeptide to generate one or more first modified polypeptides and/or one or more second modified polypeptide, respectively.

[00368] Embodiment 71. The method of embodiment 69 or 70, wherein the one or more characteristics of the sample comprises a number of polypeptides in the sample, type of polypeptides in the sample, an origin of the sample, impurities in the sample, presence of a polypeptide, absence of a polypeptide, or any combination thereof.

[00369] Embodiment 72. The method of any one of embodiments 69-71, wherein the first degradation agent and/or the second degradation agent comprises a photo-cleavable moiety. [00370] Embodiment 73. The method of any one of embodiments 69-72, wherein the first degradation agent and/or the second degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein: LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety. [00371] Embodiment 74. The method of embodiment 73, wherein LG is -SCL'.

[00372] Embodiment 75. The method of embodiment 73 or 74, wherein R¹ is hydrogen.

[00373] Embodiment 76. The method of any one of embodiments 73-75, wherein R² is hydrogen.

[00374] Embodiment 77. The method of any one of embodiments 69-76, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises one or more aromatic groups.

[00375] Embodiment 78. The method of any one of embodiments 69-76, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises a nitro-substituted benzyl group.

[00376] Embodiment 79. The method of any one of embodiments 69-76, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety is

[00377] Embodiment 80. The method of any one of embodiments 69-79, wherein the first degradation agent and/or the second degradation agent is

[00378] Embodiment 81. The method of any one of embodiments 69-80, wherein the degradation agent comprises a photo-protected guanidinylation reagent.

[00379] Embodiment 82. The method of any one of embodiments 69-81, further comprising repeating (a), (b), and (c) one or more times to detect one or more subsequent terminal amino acids of the first polypeptide and/or the second polypeptide.

[00380] Embodiment 83. The method of any one of embodiments 69-82, wherein the first polypeptide is coupled to a first biomolecule and/or the second polypeptide is coupled to a second biomolecule.

[00381] Embodiment 84. The method of any one of embodiments 69-83, wherein (1) a first probe of the first one or more probes is coupled to a terminal amino acid of the first polypeptide and/or (2) a second probe of the second one or more probes is coupled to a terminal amino acid of the second polypeptide.

[00382] Embodiment 85. The method of embodiment 84, wherein the terminal amino acid of the first polypeptide is an N-terminal amino acid and/or the terminal amino acid of the second polypeptide is another N-terminal amino acid.

[00383] Embodiment 86. The method of any one of embodiments 69-83, wherein (1) a first probe of the first one or more probes is coupled to an internal amino acid of the first polypeptide and/or (2) a second probe of the second one or more probes is coupled to an internal amino acid of the second polypeptide.

[00384] Embodiment 87. The method of embodiment 86, wherein the first probe of the first one or more probes and/or the second probe of the second one or more probes exhibits different spectral properties when conjugated to different amino acids.

[00385] Embodiment 88. The method of any one of embodiments 84-87, wherein the first probe and/or the second probe comprises a dipyrromethene-BF2 derivative.

[00386] Embodiment 89. The method of any one of embodiments 84-88, wherein the first probe and/or the second probe exhibits different fluorescent spectral properties when conjugated to different terminal amino acids.

[00387] Embodiment 90. The method of any one of embodiments 84-89, wherein the first probe of the first one or more probes and/or the second probe of the second one or more probes is specific to an amino acid type.

[00388] Embodiment 91. The method of embodiment 90, wherein the first probe and/or the second probe comprises caseinolytic protease adaptor S (ClpS) or a modified ClpS.

[00389] Embodiment 92. The method of any one of embodiments 84-91, wherein the first probe and/or the second probe is covalently coupled to the first polypeptide and/or the second polypeptide, respectively.

[00390] Embodiment 93. The method of any one of embodiments 84-92, wherein the first probe and/or the second probe is coupled to hydroxyl, a carboxylic, an amino, or a thiol group of an amino acid of the first polypeptide and/or the second polypeptide, respectively.

[00391] Embodiment 94. The method of any one of embodiments 69-93, wherein (b) and/or (c) comprises determining (1) a first property of at least a portion of the first polypeptide and/or (2) a second property of at least a portion of the second polypeptide.

[00392] Embodiment 95. The method of any one of embodiments 69-94, wherein the one or more signals or signal change from the first one or more probes and/or the one or more signals or signal change from the second one or more probes comprises one or more fluorescent spectral properties.

[00393] Embodiment 96. The method of embodiment 94 or 95, further comprising using at least i) the one or more signals or signal change from the first one or more probes or the second one or more probes and/or ii) the removed at least one amino acid from the first polypeptide and from the second polypeptide to identify one or more characteristics of the at least the portion of the first polypeptide or the at least the portion of the second polypeptide.

[00394] Embodiment 97. The method of any one of embodiments 69-96, wherein the detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes comprises detecting one or more spectral properties of a probe conjugated to a terminal amino acid.

[00395] Embodiment 98. The method of any one of embodiments 69-97, wherein the detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes comprises stochastic optical reconstruction microscopy (STORM).

[00396] Embodiment 99. The method of any one of embodiments 69-98, wherein the detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes comprises detecting fluorescence of the probe.

[00397] Embodiment 100. The method of any one of embodiments 69-99, wherein the detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes comprises detecting fluorescence emission intensity, polarity, anisotropy, photon count, peak area, peak shape, emission spectra, lifetime, or any combination thereof.

[00398] Embodiment 101. The method of any one of embodiments 69-100, wherein the detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes is at a single wavelength.

[00399] Embodiment 102. The method of any one of embodiments 69-101, wherein detecting (1) one or more signals or signal change from the first one or more probes and/or (2) one or more signals or signal change from the second one or more probes is at a plurality of wavelengths.

[00400] Embodiment 103. The method of any one of embodiments 69-102, wherein the first polypeptide and/or the second polypeptide is coupled to a surface or support. [00401] Embodiment 104. The method of embodiment 103, wherein the support comprises a bead, a polymer matrix, a membrane, an array, a microscopic slide, a glass surface, a plastic surface, a transparent surface, hydrogel, a metallic surface, a metal surface, a magnetic surface, a well, a multi-well plate, a planar surface, a nanoparticle, a microparticle, a lantern, a nanomaterial, a resin, a functionalized surface, or any combination thereof.

[00402] Embodiment 105. The method of any one of embodiments 69-104, wherein the terminus of the first polypeptide and/or the second polypeptide is coupled to the surface or support.

[00403] Embodiment 106. The method of any one of embodiments 69-105, wherein the sample is a biological sample.

[00404] Embodiment 107. The method of embodiment 106, wherein the biological sample comprises biological fluid, a tissue sample, a cell culture, a cell, or a portion thereof.

[00405] Embodiment 108. The method of any one of embodiments 69-107, wherein the first degradation agent and/or the second degradation agent comprise the same chemical structure.

[00406] Embodiment 109. The method of any one of embodiments 69-108, wherein the first degradation agent and/or the second degradation agent comprise the different chemical structures.

[00407] Embodiment 110. The method of any one of embodiments 69-109, wherein the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength from 200 nm to 750 nm.

[00408] Embodiment 111. The method of any one of embodiments 70-110, wherein the contacting comprises contacting the terminus of the first polypeptide with the first degradation agent and/or the terminus of the second polypeptide with the second degradation agent with condition sufficient to generate the first modified polypeptide and/or the second modified polypeptide.

[00409] Embodiment 112. The method of embodiment 111, wherein the condition sufficient to generate the first modified polypeptide and/or the second modified polypeptide comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof. [00410] Embodiment 113. The method of embodiment 111, wherein the condition comprises a light source and/or an aqueous basic buffer.

[00411] Embodiment 114. The method of embodiment 113, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0. [00412] Embodiment 115. The method of embodiment 111, wherein the condition sufficient to generate the first modified polypeptide and/or second modified polypeptide comprises a reaction temperature from about 30 degrees to 60 degrees.

[00413] Embodiment 116. The method of any one of embodiments 69-115, further comprising identifying a terminal amino acid of the first polypeptide and/or the second polypeptide by comparing spectral properties of the first one or more probes and/or the second one or more probes to a plurality of reference spectral properties.

[00414] Embodiment 117. The method of embodiment 116, wherein a spectral property of the spectral properties is representative of a probe conjugated to a different terminal amino acid.

[00415] Embodiment 118. The method of any one of embodiments 69-117, wherein the first polypeptide and/or the second polypeptide comprises one or more natural amino acids.

[00416] Embodiment 119. The method of any one of embodiments 69-118, wherein the first polypeptide and/or the second polypeptide comprises one or more unnatural amino acids.

[00417] Embodiment 120. A compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

[00418] Embodiment 121. The compound of embodiment 120, wherein LG is -SCh'.

[00419] Embodiment 122. The compound of embodiment 120 or 121, wherein R¹ is C1-6 alkyl or hydrogen.

[00420] Embodiment 123. The compound of any one of embodiments 120-122, wherein R² is C1-6 alkyl or hydrogen.

[00421] Embodiment 124. The compound of any one of embodiments 120-123, wherein each of R¹ and R² are hydrogen.

[00422] Embodiment 125. The compound of any one of embodiments 120-124, wherein the photo-cleavable moiety comprises one or more aromatic groups. [00423] Embodiment 126. The compound of any one of embodiments 120-125, wherein the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of about 200 nm to about 750 nm.

[00424] Embodiment 127. The compound of any one of embodiments 120-126, wherein the photo-cleavable moiety comprises a nitro- substituted benzyl group.

[00425] Embodiment 128. The compound of any one of embodiments 120-126, wherein the photo-cleavable moiety is

[00426] Embodiment 129. The compound of any one of embodiments 120-126, wherein the degradation agent is

[00427] Embodiment 130. The compound of any one of embodiments 120-129, wherein the degradation agent is

[00428] Embodiment 131. A method, comprising: (a) providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes; (b) detecting one or more signals or signal change from the one or more probes; (c) subjecting at least a portion of the polypeptide to (1) a first light comprising a first one or more wavelengths sufficient to remove the one or more probes, and (2) second light comprising a second one or more wavelengths sufficient to remove a terminal amino acid of the polypeptide.

[00429] Embodiment 132. The method of embodiment 131, repeating (a) and (b) one or more additional times on one or more subsequent amino acids of the polypeptide to generate one or more additional modified polypeptides.

[00430] Embodiment 133. The method of embodiment 131 or 132, wherein (c) comprises contacting a terminus of the polypeptide with a degradation agent.

[00431] Embodiment 134. The method of embodiment 133, wherein the degradation agent comprises a photo-cleavable moiety.

[00432] Embodiment 135. The method of any one of embodiments 131-134, wherein the first light is a different light compared to the second light.

[00433] Embodiment 136. The method of any one of embodiments 131-135, wherein the first one or more wavelength and/or the second one or more wavelengths is from 200 nm to 500 nm. [00434] Embodiment 137. The method of any one of embodiments 131-136, wherein a probe of the one or more probes is removed prior to removal of the terminal amino acid of the polypeptide.

[00435] Embodiment 138. The method of any one of embodiments 131-136, wherein a probe of the one or more probes is removed subsequent to removal of the terminal amino acid of the polypeptide.

[00436] Embodiment 139. The method of any one of embodiments 131-138, further comprising contacting a terminus of the polypeptide with a degradation agent, thereby forming a first modified polypeptide, comprising a photo-cleavable moiety.

[00437] Embodiment 140. The method of any one of embodiments 131-139, subjecting the first modified polypeptide to a condition sufficient to generate a second modified polypeptide and a fragment comprising a residue of the terminus of the polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

[00438] Embodiment 141. The method of any one of embodiments 131-140, further comprising determining at least one characteristic of the at least a portion of the polypeptide. [00439] Embodiment 142. The method of any one of embodiments 131-141, wherein the detecting of (b) occurs prior to (c).

[00440] Embodiment 143. The method of any one of embodiments 131-142, wherein the detecting of (b) occurs subsequent to (c). [00441] Embodiment 144. The method of any one of embodiments 134-143, wherein the photo-cleavable moiety comprises one or more aromatic groups.

[00442] Embodiment 145. The method of any one of embodiments 134-143, wherein the photo-cleavable moiety comprises a nitro- substituted benzyl group.

[00443] Embodiment 146. The method of any one of embodiments 134-143, wherein the photo-cleavable moiety is

[00444] Embodiment 147. The method of any one of embodiments 131-146, wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids.

[00445] Embodiment 148. The method of embodiment 147, wherein the probe comprises a dipyrromethene-BF2 derivative.

[00446] Embodiment 149. The method of any one of embodiments 131-146, wherein a probe of the one or more probes is specific to an amino acid type.

[00447] Embodiment 150. The method of any one of embodiments 131-149, further comprising using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide.

[00448] Embodiment 151. The method of any one of embodiments 131-150, wherein the detecting the one or more signals or signal change comprises detecting one or more spectral properties for each probe conjugated to a terminal amino acid.

[00449] Embodiment 152. The method of any one of embodiments 131-151, wherein the detecting the one or more signals or signal change comprises stochastic optical reconstruction microscopy (STORM).

[00450] Embodiment 153. The method of any one of embodiments 131-152, wherein the polypeptide is coupled to a surface or support.

[00451] Embodiment 154. The method of any one of embodiments 131-153, wherein the polypeptide is among a sample comprising a plurality of analytes.

[00452] Embodiment 155. The method of embodiment 154, wherein the sample is a biological sample.

[00453] Embodiment 156. The method of embodiment 154 or 155, wherein an analyte of the plurality of analytes comprises a polypeptide. [00454] Embodiment 157. The method of any one of embodiments 131-156, further comprising providing one or more additional polypeptides comprising one or more probes coupled to one or more amino acids of the one or more polypeptides.

[00455] Embodiment 158. The method of embodiment 157, further comprising contacting one or more additional polypeptides with one or more additional degradation agents, thereby forming one or more additional modified polypeptides.

[00456] Embodiment 159. The method of embodiment 157 or 158, wherein the one or more additional modified polypeptides comprises one or more additional photo-cleavable moieties.

[00457] Embodiment 160. The method of embodiments 158 or 159, further comprising subjecting the one or more additional modified polypeptides to conditions sufficient to generate one or more cleaved polypeptides.

[00458] Embodiment 161. The method of any one of embodiments 158-160, wherein the degradation agent and the one or more additional degradation agents comprise the same chemical structure.

[00459] Embodiment 162. The method of any one of embodiments 140-161, wherein the condition sufficient to generate the second modified polypeptide comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent or any combination thereof.

[00460] Embodiment 163. The method of any one of embodiments 131-162, further comprising identifying a terminal amino acid of the polypeptide by comparing spectral properties of the probe to a plurality of reference spectral properties.

[00461] Embodiment 164. A method, comprising: (a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide; and (b) subjecting at least a portion of the first modified polypeptide in a solution to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the solution comprises one or more acids, wherein the second modified polypeptide comprises one or more fewer amino acids than the polypeptide.

[00462] Embodiment 165. The method of embodiment 164, wherein the degradation agent comprises a photo-cleavable moiety.

[00463] Embodiment 166. The method of embodiment 164 or 165, wherein the one or more acids comprises a Lewis acid.

[00464] Embodiment 167. The method of embodiment 166, wherein the Lewis acid comprises scandium tritiate, ytterbium tritiate, zinc tritiate, or any combination thereof. [00465] Embodiment 168. The method of any one of embodiments 164-167, wherein pH of the solution is from 7 to 13.

[00466] Embodiment 169. The method of any one of embodiments 164-168, wherein the solution comprises the one or more acids at a concentration from 0.1 M to 1 M.

[00467] Embodiment 170. The method of any one of embodiments 164-169, wherein the light comprises a wavelength from 200 nm to 500 nm.

[00468] Embodiment 171. The method of any one of embodiments 165-170, wherein the photo-cleavable moiety comprises one or more aromatic groups.

[00469] Embodiment 172. The method of any one of embodiments 165-170, wherein the photo-cleavable moiety comprises a nitro- substituted benzyl group.

[00470] Embodiment 173. The method of any one of embodiments 165-170, wherein the photo-cleavable moiety is

[00471] Embodiment 174. The method of any one of embodiments 165-173, wherein, prior to (a), the method further comprises providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes.

[00472] Embodiment 175. The method of embodiment 174, wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids. [00473] Embodiment 176. The method of embodiment 175, wherein the probe comprises a dipyrromethene-BF2 derivative.

[00474] Embodiment 177. The method of embodiment 174, wherein a probe of the one or more probes is specific to an amino acid type.

[00475] Embodiment 178. The method of any one of embodiments 164-177, further comprising detecting one or more signals or signal change from the one or more probes to identify a characteristic of at least a portion of the polypeptide.

[00476] Embodiment 179. The method of any one of embodiments 164-178, further comprising using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide. [00477] Embodiment 180. The method of embodiment 178 or 179, wherein the detecting the one or more signals or signal change comprises detecting one or more spectral properties for each probe conjugated to a terminal amino acid. [00478] Embodiment 181. The method of any one of embodiments 178-180, wherein the detecting the one or more signals or signal change comprises stochastic optical reconstruction microscopy (STORM).

[00479] Embodiment 182. The method of any one of embodiments 164-181, wherein the polypeptide is coupled to a surface or support.

[00480] Embodiment 183. The method of any one of embodiments 164-182, wherein the polypeptide is among a sample comprising a plurality of analytes.

[00481] Embodiment 184. The method of embodiment 183, wherein the sample is a biological sample.

[00482] Embodiment 185. The method of embodiment 183 or 184, wherein an analyte of the plurality of analytes comprises the polypeptide.

[00483] Embodiment 186. The method of any one of embodiments 164-185, further comprising providing one or more additional polypeptides comprising one or more probes coupled to one or more amino acids of the one or more polypeptides.

[00484] Embodiment 187. The method of embodiment 186, further comprising contacting the one or more additional polypeptides with one or more additional degradation agents, thereby forming one or more additional modified polypeptides.

[00485] Embodiment 188. The method of embodiment 187, wherein the one or more additional modified polypeptides comprises one or more additional photo-cleavable moieties.

[00486] Embodiment 189. The method of embodiment 187 or 188, further comprising subjecting the one or more additional modified polypeptides to conditions sufficient to generate one or more cleaved polypeptides.

[00487] Embodiment 190. The method of any one of embodiments 186-189, wherein the degradation agent and the one or more additional degradation agents comprise the same chemical structure.

[00488] Embodiment 191. The method of embodiment 189 or 190, wherein the condition sufficient to generate the one or more cleaved peptides comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof.

[00489] Embodiment 192. The method of any one of embodiments 164-191, further comprising identifying a terminal amino acid of the polypeptide by comparing spectral properties of the probe to a plurality of reference spectral properties.

[00490] Embodiment 193. A method for analyzing a sample, comprising: (a) providing a first polypeptide to a first location on a first support and a second polypeptide to a second location on a second support, wherein the first polypeptide is coupled to first one or more probes; (b) detecting one or more signals or signal change from the one or more probes of the first polypeptide; (c) selectively subjecting at least a portion of the first polypeptide to light comprising one or more wavelengths sufficient to selectively remove a terminal amino acid of the first polypeptide.

[00491] Embodiment 194. The method of embodiment 193, Repeating (b) and (c) one or more additional times on one or more subsequent amino acids of the first polypeptide to generate one or more additional modified polypeptides.

[00492] Embodiment 195. The method of embodiment 193 or 194, wherein (c) comprises contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent.

[00493] Embodiment 196. The method of any one of embodiments 193-195, wherein the first degradation agent and/or the second degradation agent comprises a photo-cleavable moiety.

[00494] Embodiment 197. The method of any one of embodiments 193-196, wherein (c) comprises subjecting the at least the portion of the first polypeptide to a first degradation agent to selectively remove the terminal amino acid of the first polypeptide.

[00495] Embodiment 198. The method of any one of embodiments 193-197, wherein (c) comprises providing the light to the at least the portion of the first polypeptide at the first location of the first support.

[00496] Embodiment 199. The method of any one of embodiments 193-198, wherein (c) comprises providing the light to the at least the portion of the first polypeptide at the first location of the first support, but not to at least a portion of the second polypeptide at the second location of the second support.

[00497] Embodiment 200. The method of any one of embodiments 193-199, wherein the selectively subjecting comprises (1) subjecting the at least the portion of the first polypeptide to the light at a first time and (2) subjecting at least a portion of the second polypeptide to another light at a second time.

[00498] Embodiment 201. The method of embodiment 200, wherein the second time is subsequent to the first time.

[00499] Embodiment 202. The method of any one of embodiments 193-201, wherein the first support is the same support as the second support.

[00500] Embodiment 203. The method of any one of embodiments 193-202, wherein a distance between the first location and the second location is at most about 10 cm. [00501] Embodiment 204. The method of any one of embodiments 193-203, wherein a distance between the first location and the second location is at least about 150 nm.

[00502] Embodiment 205. The method of any one of embodiments 193-204, wherein the first degradation agent and/or the second degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein: LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety.

[00503] Embodiment 206. The method of embodiment 205, wherein LG is -SCL'.

[00504] Embodiment 207. The method of embodiment 205 or 206, wherein R¹ is hydrogen.

[00505] Embodiment 208. The method of any one of embodiments 205-207, wherein R² is hydrogen.

[00506] Embodiment 209. The method of any one of embodiments 193-208, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises one or more aromatic groups.

[00507] Embodiment 210. The method of any one of embodiments 193-209, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises a nitro-substituted benzyl group.

[00508] Embodiment 211. The method of any one of embodiments 193-210, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety is

[00509] Embodiment 212. The method of any one of embodiments 193-211, wherein the first degradation agent and/or the second degradation agent is [00510] Embodiment 213. The method of any one of embodiments 193-212, wherein the second polypeptide is coupled to second one or more probes.

[00511] Embodiment 214. The method of any one of embodiments 193-213, wherein (1) a first probe of the first one or more probes is coupled to a terminal amino acid of the first polypeptide and/or (2) a second probe of the second one or more probes is coupled to a terminal amino acid of the second polypeptide.

[00512] Embodiment 215. The method of any one of embodiments 193-213, wherein (1) a first probe of the first one or more probes is coupled to an internal amino acid of the first polypeptide and/or (2) a second probe of the second one or more probes is coupled to an internal amino acid of the second polypeptide.

[00513] Embodiment 216. The method of any one of embodiments 193-215, wherein a first probe of the first one or more probes and/or a second probe of the second one or more probes exhibits different spectral properties when conjugated to different amino acids.

[00514] Embodiment 217. The method of any one of embodiments 193-216, wherein a first probe of the first one or more probes and/or a second probe of the second one or more probes is specific to an amino acid type.

[00515] Embodiment 218. The method of any one of embodiments 193-217, wherein (b) and/or (c) comprises determining (1) a first property of at least a portion of the first polypeptide and/or (2) a second property of at least a portion of the second polypeptide.

[00516] Embodiment 219. The method of any one of embodiments 193-218, wherein the one or more signals or signal change comprises one or more fluorescent spectral properties.

[00517] Embodiment 220. The method of any one of embodiments 193-219, further comprising using at least i) the one or more signals or signal change and/or ii) the removed at least one amino acid from the first polypeptide and from the second polypeptide to identify the characteristic of the at least the portion of the polypeptide.

[00518] Embodiment 221. The method of any one of embodiments 193-220, wherein the detecting the one or more signals or signal change comprises detecting one or more spectral properties for each probe conjugated to a terminal amino acid.

[00519] Embodiment 222. The method of any one of embodiments 193-221, wherein the detecting the one or more signals or signal change comprises stochastic optical reconstruction microscopy (STORM).

[00520] Embodiment 223. The method of any one of embodiments 193-222, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety is cleavable when subjected to one or more conditions comprises a pH condition, an optical condition, a light source, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or any combination thereof.

[00521] Embodiment 224. The method of embodiment 223, wherein the buffer condition comprises an aqueous basic buffer.

[00522] Embodiment 225. The method of embodiment 223 or 224, wherein the one or more conditions comprises the light source and/or the aqueous basic buffer.

[00523] Embodiment 226. The method of embodiment 225, wherein the aqueous basic buffer comprises a pH value from about 9.0 to about 13.

[00524] Embodiment 227. The method of any one of embodiments 193-226, further comprising identifying a terminal amino acid of the first polypeptide and/or the second polypeptide by comparing spectral properties of the first one or more probes and/or the second one or more probes to a plurality of reference spectral properties.

[00525] Embodiment 228. A method, comprising: (a) detecting one or more signals or signal change from one or more probes coupled to a polypeptide; (b) subjecting at least a portion of the polypeptide to a light condition sufficient to remove a terminal amino acid of the polypeptide;

(c) using the one or more signals or signal change to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.

[00526] Embodiment 229. The method of embodiment 228, wherein the one or more probes is coupled to one or more amino acids of the polypeptide.

[00527] Embodiment 230. The method of embodiment 228 or 229, wherein the one or more signals or signal change determines one or more characteristics of the at least the portion of the analyte with an accuracy of at least 85%.

[00528] Embodiment 231. The method of any one of embodiments 228-230, further comprising, prior to (b), contacting a terminus of the polypeptide with a degradation agent. [00529] Embodiment 232. The method of embodiment 231, wherein the degradation agent comprises a photo-cleavable moiety.

[00530] Embodiment 233. The method of embodiment 231 or 232, wherein the degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein: LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety.

[00531] Embodiment 234. The method of embodiment 233, wherein LG is -SCE'.

[00532] Embodiment 235. The method of embodiment 233 or 234, wherein R¹ is hydrogen.

[00533] Embodiment 236. The method of any one of embodiments 233-235, wherein R² is hydrogen.

[00534] Embodiment 237. The method of any one of embodiments 233-236, wherein the photo-cleavable moiety comprises one or more aromatic groups.

[00535] Embodiment 238. The method of any one of embodiments 233-236, wherein the photo-cleavable moiety comprises a nitro- substituted benzyl group.

[00536] Embodiment 239. The method of any one of embodiments 228-238, wherein the polypeptide is coupled to a biomolecule.

[00537] Embodiment 240. The method of any one of embodiments 228-239, wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids.

[00538] Embodiment 241. The method of any one of embodiments 228-240, wherein a probe of the one or more probes is specific to an amino acid type.

[00539] Embodiment 242. The method of any one of embodiments 228-241, wherein the one or more signals or signal change comprises one or more fluorescent spectral properties.

[00540] Embodiment 243. The method of any one of embodiments 228-242, wherein the polypeptide is coupled to a surface or support.

[00541] Embodiment 244. The method of any one of embodiments 228-243, further comprising repeating (a)-(c) one or more times one or more subsequent amino acids of the polypeptide.

[00542] Embodiment 245. The method of any one of embodiments 228-244, further comprising identifying the terminal amino acid of the polypeptide by comparing spectral properties of the probe to a plurality of reference spectral properties.

EXAMPLES

[00543] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1. SYNTHETIC PROCEDURES

[00544] Unless otherwise noted, all reagents were purchased from commercial suppliers without further purification. All reactions were performed under nitrogen atmosphere, and were monitored via thin layer chromatography on plates (TLG-R100114B-323) supplied by SiliCycle, and visualized by UV light (254 nm or 365 nm). Normal phase flash chromatography was performed using SiliCycle F60 silica gel (40-63 jim, 60 A, R12030B). Reverse phase flash chromatography was performed using Biotage Sfar C18 Duo columns (100 A 30 jim, FSUD- 401) on a Advion Interchim puriFlash 5.250 using a mixture of water with 0.01% TFA and acetonitrile with 0.01% TFA. Yields refer to chromatographically and spectroscopically (NMR) pure materials. 1H NMR, 13C NMR, and 19F NMR spectra were recorded at ambient temperature on a Varian Agilent 500 MHz VNMRS (500, 126, and 470 MHz respectively), and were internally referenced to the residual protio solvent signal (CDC13: 5 7.26 and 77.16 ppm, CD3CN: 5 1.94 and 118.26 and 1.32 ppm, d-DMSO: 5 2.50 and 39.52 ppm, and D2O: 4.79 ppm). Data for 1H and 19F NMR spectra are reported as follows: chemical shift, integration, multiplicity (brs = broad singlet, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, overlap = overlapping peaks) and coupling constants in Hz and data for 13C NMR are reported in terms of chemical shift. Analytical UPLC-MS experiments were performed using a Waters Acquity (ultraperformance liquid chromatography) with a binary solvent manager, an SQ mass spectrometer, a Waters 2996 photodiode array (PDA) detector, and an evaporative lightscattering detector (ELSD). The column used was an ACQUITY UPLC BEH Cl 8 Column, 130A, 1.7 pm, 2.1 mm X 50 mm.

A. Synthesis of Nvoc-chloroformate (3.36)

[00545] In a 500 mL round bottom flask, equipped with magnetic stir bar, was added 100 mL of nitric acid. The flask was cooled under gentle stirring with a water-ice bath. To the cooled flask was added 3,4-dimethoxybenzaldehyde (3.33) (100 g, 602 mmol) portion wise. The suspension was allowed to warm to room temperature over 1 h and all the solids dissolved. The solution turned into a dark orange. Following this time, the reaction solution was poured slowly into 500 mL of ice water, and a yellow solid precipitated out. The solid was filtered, and recrystallized in ethanol to produce (3.34) (114.5 g, 542 mmol, 90% yield) as a yellow solid. [00546] (3.34) (114.5 g, 542 mmol, 1 eq) was added to a 500 mL round bottom flask equipped with magnetic stir bar. 200 mL of ethanol was added to the flask and the suspension was cooled to 0 °C with a water-ice bath. To the suspension was slowly added sodium borohydride (10.26 g, 271 mmol, 0.5 eq). A gas evolved upon addition, and the solution became fully dissolved. The reaction was allowed to stir for 1 h at room temperature. Following this time, the reaction was quenched with aq. sat. NH4C1 until the evolution of gas ceased. The solution was then concentrated under reduced pressure to remove the majority of the ethanol. The solution was then taken up in ethyl acetate (200 mL) and was washed sequentially with 1 M HC1 (200 mL), aq. sat. NaHCO3 (200 mL), and brine (200 mL). The organic layer was dried with sodium sulfate, and dried under reduced pressure to give (3.35) (115 g, 542 mmol, >99% yield) as a yellow solid.

[00547] (3.35) (10 g, 47 mmol, 1 eq) was added to a flame dried 250 mL round bottom flask equipped with magnetic stir bar and rubber septum under inert atomosphere. Anhydrous dichloromethane (100 mL) was added to the flask followed by triphosgene (14 g, 47 mmol, 1 eq). Tri ethylamine (6.5 mL, 47 mmol, 1 eq) was added dropwise to the solution. Upon full addition, the reaction was wrapped in foil to remove from light, and was allowed to stir under inert atmosphere at room temperature for 16 h. Following this time, the reaction was diluted with ethyl acetate (200 mL), and washed with sequentially with 1 M HC1 (200 mL), aq. sat. NaHCO3 (200 mL), and brine (200 mL). The organic layer was dried with sodium sulfate, and dried under reduced pressure to give (3.36) (11.1 g, 47 mmol, 86% yield) as a yellow solid.

4.5-dimethoxy-2-nitrobenzaldehyde (3.34)

90% yield, yellow solid

1H NMR: (500 MHz, CDC13) 5 10.45 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 4.04 (s, 3H), 4.03 (s, 3H).

13C NMR: (126 MHz, CDC13) 5 187.73, 153.22, 152.40, 125.57, 109.78, 107.18, 56.82, 56.77. (4,5-dimethoxy-2-nitrophenyl) methanol (3.35) >99% yield, yellow solid

1HNMR: (500 MHz, CDC13) 5 7.71 (s, 1H), 7.17 (s, 1H), 4.97 (s, 2H), 4.01 (s, 3H), 3.96 (s, 3H), 2.59 (s, 1H).

13C NMR: (126 MHz, CDC13) 5 153.89, 132.21, 111.06, 108.13, 62.89, 56.48, 56.40.

4.5-dimethoxy-2-nitrobenzyl carbonochloridate (3.36) 86% yield, yellow solid

1H NMR: (500 MHz, CDC13) 5 7.76 (s, 1H), 7.01 (s, 1H), 5.73 (d, J = 0.7 Hz, 2H), 4.01 (s, 3H), 3.98 (s, 3H).

13C NMR: (126 MHz, CDC13) 5 153.73, 150.39, 148.91, 139.86, 124.23, 110.37, 108.36, 69.83, 56.55, 56.50. B. Synthesis of Nvoc-pentafluorophenol carbonate (3.39)

[00548] (3.35) (10 g, 47 mmol, 1 eq) and bis(pentafluoropheynl)carbonate (3.38) (22 g, 56 mmol, 1.2 eq) were added to a 250 mL round bottom flask equipped with magnetic stir bar. To the flask was added dichloromethane (100 mL) followed by diisopropylethylamine (12.3 mL, 56 mL, 1.2 eq). Upon full addition, the reaction was wrapped in foil to remove from light, and was allowed to stir at room temperature for 16 h. Following this time, the reaction was diluted with ethyl acetate (200 mL), and washed with sequentially with 1 M HC1 (200 mL), aq. sat. NaHCO3 (200 mL), and brine (200 mL). The organic layer was dried with sodium sulfate, and dried under reduced pressure to give (3.39) (20 g, 47 mmol, 99% yield) as a yellow oil.

[00549] 1H NMR: (500 MHz, CDC13) 5 7.78 (s, 1H), 7.07 (s, 1H), 5.77 (s, 2H), 4.03 (s, 3H), 3.98 (s, 3H).

[00550] 13C NMR: (126 MHz, CDC13) 5 153.93, 151.05, 148.75, 139.65, 138.94, 136.92,

125.19, 109.48, 108.42, 77.26, 77.00, 76.75, 68.64, 56.46.

[00551] 19F NMR: (470 MHz, CDC13) 5 -153.30 - -153.44 (m), - 156.86 (t, J = 21.7 Hz), -

161.64 (dd, J = 22.5, 17.7 Hz), - 163.31 - -163.42 (m), -163.97 (td, J = 22.5, 22.0, 4.6 Hz), - 168.66 - -168.82 (m).

C. Synthesis of degrader (3.32)

[00552] Methyl carbamimidothioate sulfate (3.37) (500 mg, 2.66 mmol, 1 eq) and Nvoc-PFP (3.39) (4.50 g, 10.6 mmol, 4 eq) were added to a 50 mL round bottom flask equipped with magnetic stir bar followed by acetonitrile (20 mL). Diisopropylethylamine (2.78 mL, 15.9 mmol, 6 eq) was added and the solution was wrapped in foil to remove from light, and was allowed to stir at room temperature for 16 h. Following this time, the reaction was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with sequentially with 1 M HC1 (100 mL), aq. sat. NaHCO3 (100 mL), and brine (100 mL). The organic layer was dried with sodium sulfate, and dried under reduced pressure. The crude solid was purified by flash chromatography (20% EtOAc/Hexanes) to give (3.32) (530 mg, 0.93 mmol, 35% yield) as a yellow oil.

D. Synthesis of amidine sulfonates (3.50)

[00553] Following literature procedure, 34 substituted thiourea (3.49) (5.55 mmol, 1 eq) was dissolved in 1: 1 acetonitrile/water. The solution was cooled to -15 °C with a brine-ice bath.

Hydrogen peroxide (30% w/v, 16.6 mmol, 3 eq) was added dropwise to the solution which caused rigorous bubbling. After full addition of the hydrogen peroxide, the solution was allowed to warm to room temperature overnight. The reaction was filtered, and concentrated under reduced pressure. The crude solid was then recrystallized in acetonitrile, filtered, and dried to give the final amidine sulfonate (3.50).

N-methylamino(imino) sulfonic acid (3.52)

86% yield, white solid

1H NMR: (500 MHz, D2O) 5 3.35 (d, J = 4.2 Hz, 1H), 2.90 (d, J = 4.3 Hz, 3H), 2.79 (d, J = 4.5 Hz, 1H).

13C NMR: (126 MHz, D2O) 5 177.49, 164.15, 154.73, 31.53, 28.94, 28.65.

N-ethylamino(imino) sulfonic acid (3.53)

23% yield, white solid 'H NMR: (500 MHz, d-DMSO) 5 9.56 (t, J = 6.4 Hz, 1H), 9.17 (s, 1H), 9.09 (s, 1H), 3.23 (p, J = 6.9 Hz, 2H), 1.07 (t, J = 7.2 Hz, 3H).

¹³C NMR: (126 MHz, d-DMSO) 5 165.54, 40.28, 40.11, 39.95, 39.78, 39.61, 39.45, 39.28, 37.61,

13.21.

N-benzylamino(imino) sulfonic acid (3.56) -DMSO) 5 9.41 (s, 1H), 9.04-8.78 (m,lH), 7.36 (t, J = 7.3 Hz, 2H), 7.30 (d, d,J = 10.3 Hz, 2H). d-DMSO) 5 136.48, 128.98, 128.04,127.91, 127.72, 45.09. thylimino) sulfonic acid (3.57)

74% yield, clear oil

‘H NMR: (500 MHz, D2O) 5 3.12 (d, J = 5.0 Hz, 3H), 2.80 (t, J = 3.4 Hz, 3H).

¹³C NMR: (126 MHz, D2O) 5 165.65, 162.33, 155.99, 38.45, 34.75, 32.86, 31.01, 28.56, 27.65,

27.21, 26.92, 26.63, 24.70.

N, N-dimethylamino(imino) sulfonic acid (3.58)

17% yield, white solid

'H NMR: (500 MHz, d-DMSO) 5 8.92 (s, 1H), 3.25 (d, J = 147.7 Hz, 6H).

¹³C NMR: (126 MHz, d-DMSO) 5 165.69, 41.03.

4, 5-dihydro-lH-imidazole-2-sulfonate (3.59) 47% yield, white solid

’H NMR: (500 MHz, d-DMSO) 5 10.19 (s, 2H), 3.85 (s, 4H).

¹³C NMR: (126 MHZ, d-DMSO) 5 170.15, 45.38.

E. Synthesis of Nvoc Protected Amidine Sulfonates (3.51)

(3.50) (3.51)

[00554] In a 20 mL scintillation vial, equipped with magnetic stir bar, was added Nvoc-PFP (3.39) (500 mg, 1.18 mmol, 1 eq) and amidine sulfonate (1.77 mmol, 1.5 eq). Acetonitrile (5 mL) and aq. sat. NaHCO3 (5 mL) were added to the vial, and the solution was allowed to stir under darkness at room temperature overnight. After this time, the reaction was concentrated under reduced pressure. The crude mixture was then triturated with ethyl acetate (3 x 10 mL) to remove organic byproducts. The precipitant was then taken up in methanol, filtered, and dried under reduced pressure. The residue was then taken up in water and purified via reverse phase liquid chromatography from 0-20% ACN/water without exposure to light to yield the final product (3.51).

((((4,5-dimethoxy-2-nitrobenzyl)oxy)carbonyl)amino)(imino)methanesulfonate (3.45)

59% yield, yellow solid

'H NMR: (500 MHz, d-DMSO) 7.67 (d, J = 3.9 Hz, 1H), 7.23 (d, J = 13.8 Hz, 1H), 5.33 (d, J = 10.2 Hz, 2H), 3.94 - 3.80 (m, 6H).

¹³C NMR: (126 MHz, d-DMSO) 5 165.43, 156.63, 154.28, 154.04, 153.96, 148.29, 127.78, 127.59, 111.88, 108.56, 63.99, 63.77, 63.54, 62.44, 56.90, 56.75, 56.63, 56.54, 54.37, 45.87, 40.48, 40.31, 40.15, 39.98, 39.81, 39.65, 39.48, 9.19, 9.16.

(Z)-((((4,5-dimethoxy-2-nitrobenzyl)oxy)carbonyl)amino)(methylimino)methanesulfonate (3.60)

35% yield, yellow solid 'H NMR: (500 MHz, d-DMSO) 5 7.66 (s, 1H), 7.59 (s, 1H), 7.36 (s, 1H), 5.31 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 3.03 (d, J = 7.4 Hz, 3H).

¹³C NMR: (126 MHz, d-DMSO) 5 178.68, 176.21, 166.65, 154.21, 40.31, 40.15, 40.08, 39.98, 39.81, 39.79, 39.65, 39.48, 39.31, 31.94, 29.52, 29.37, 29.25, 29.11.

(Z)-((((4, 5-dimethoxy-2-nitrobenzyl)oxy)carbonyl )amino)(ethylimino)methanesulfonate (3.61)

22% yield, yellow solid

‘H NMR: (500 MHz, d-DMSO) 5 7.65 (s, 1H), 7.59 (s, 1H), 7.37 (s, 1H), 5.31 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 3.10 (q, J = 6.7 Hz, 2H), 1.06 (t, J = 7.2 Hz, 3H). ¹³C NMR: (126 MHz, d- DMSO) 5 160.34, 158.23, 154.17, 147.77, 139.18, 129.44, 111.54, 108.66, 108.11, 63.54, 57.10, 56.48, 46.28, 40.54, 40.37, 40.21, 40.04, 39.88, 39.71, 39.54, 36.30, 14.07, 9.29, 9.27.

(Z)-((((4, 5 -dime thoxy-2- nitrobenzyl)oxy)carbonyl )(methyl)amino)(methylimino)methanesulfonate (3.63) soy N' .

19% yield, yellow solid

‘H NMR: (500 MHz, d-DMSO) 5 7.65 (s, 1H), 7.38 (s, 1H), 4.82 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 2.49 (t, J = 1.9 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H), 1.07 (t, J = 7.2 Hz, 3H).

¹³C NMR: (126 MHZ, d-DMSO) 5 153.99, 147.45, 134.58, 110.17, 108.35, 60.55, 58.20, 56.71, 56.55, 56.52, 56.50, 46.18, 40.54, 40.37, 40.33, 40.21, 40.04, 39.87, 39.71, 39.54, 8.15.

EXAMPLE 2. PHOTO-CAGED AMIDINE FOR N-TERMINAL DEGRADATION OF MODEL PROTEINS

[00555] A degradation agent (3.45) was tested with the model protein angiotensin I (3.46) and observed conjugation to the N-terminus (3.47) within 30 minutes in basic aqueous buffer without the use of organic cosolvents (FIG. 4). Following the conjugation, N-degradation of the conjugated species (3.47) occurred rapidly upon irradiation in basic aqueous media, forming the degraded angiotensin I peptide fragment (3.48). This represented a marked improvement over initial degradation agent in both water solubility and reactivity, demonstrating that amidine sulfonates as guanidinylation reagents were worth further investigation. Additionally, it was observed that no N-degradation of the conjugated species (3.47) when not exposed to light, even in higher pH buffers, suggesting that the hypothesis regarding the single carbamate was sufficient to quell the nucleophilicity of the photocaged amidine.

[00556] To further test the kinetics of the initial N-conjugation with the degradation agent

(3.45), a time series with degradation agent (3.45) and amine-bearing silica beads was performed. In this assay, these amine beads were incubated with (3.45) in bicarbonate buffer for 65 minutes and took time points at 5-minute increments by removing an aliquot of beads and washing away the remaining unreacted degradation agent. At the end of the 65 minutes, all beads were then reacted with AlexaFluor488-NHS, an amine-reactive fluorescent dye, and the overall fluorescent signal for each bead was obtained for each time point (FIG. 5).

[00557] From this assay, it was observed that all available amines have reacted with degradation agent (3.45) within 45 minutes, where the signal drops to near zero and remains there for the remainder of the assay. Additionally, it was observed that fluorescent quenching of the AlexaFluor in the early time points, where the signal is low and progressively rises at 25 minutes. This assay demonstrates that the photocaged amidine sulfonate degradation agent

(3.45) reacts quickly and efficiently with amines in aqueous buffer, even when bound to a surface such as silica.

EXAMPLE 3. STRUCTURE ACTIVITY RELATIONSHIP OF PHOTOCAGED AMIDINE SULFONATES

[00558] Following the promising results from the photocaged amidine sulfonate degradation agent (3.45), which displayed excellent N-terminal conjugation kinetics and water solubility, attempts were made to optimize the N-degradation kinetics as well. While the degrader for solubility and N-terminal conjugation reactivity may be optimized, the core N-terminal guanidine that is formed has been shown to exhibit significantly slower N-degradation kinetics in buffers with a pH less than 13 (FIG. 6). It is aimed to develop a degradation agent capable of guanidinylating and cleaving the N-terminal amino acid successfully and quickly at pH 9-10, as higher pH buffers may affect protein stability. It is believed that substituting one or both nitrogens on the amidine with increasing steric bulk may enhance the rate of cleavage, as the steric repulsion between the amino acid side chain and the amidine substitution may further drive the nucleophilic nitrogen toward the amide carbonyl via a Thorpe-Ingold effect (FIG. 7). [00559] A small library of amidine sulfonic acids (3.50) was developed by oxidizing their corresponding thioureas (3.49) and subsequently forming the photocaged amidines (3.51) with Nvoc-PFP (3.39) (FIG. 8). [00560] The thioureas chosen for this structure-activity library were selected with increasing substituents on one or both nitrogens. By increasing the steric bulk substituted on the final photocaged amidine, how substitution affects both N-terminal degradation rates and N-terminal conjugation was understood.

[00561] All sulfonates formed through oxidation were able to generate the final photocaged amidine degradation agent in appreciable amounts (FIG. 8). However, it is observed that the oxidation of //-propyl and /-propyl substituted thioureas was unsuccessful. It is believed this may be due to the self-assembly of the oxidized forms of these thioureas, forming micellar structures. These micelles may have made the purification of the final amidine sulfonate difficult. NMRs for each of analogues are shown in FIGs. 9A-9H.

[00562] The library was first tested against the unsubstituted degradation agent (3.45) for their ability to react with a model peptide, angiotensin 1 (FIG. 10). Unfortunately, the addition of substitutions significantly slowed down the conjugation reaction kinetics, with degradation agent (3.62) only partially reacting after 16 hours and the disubstituted degradation agents (3.63), (3.64), and (3.65) not reacting at all. Monosubstituted degradation agents (3.60) and (3.61) did fully conjugate with angiotensin 1, but still at a significantly reduced rate compared to the unsubstituted (3.45). While it was expected that the N-terminal conjugation reaction kinetics to slow down, it was not anticipated that the kinetics may drop so precipitously with all substitutions.

[00563] The conjugated angiotensin 1 species were then purified and subjected to two different degradation buffers with irradiation: pH 9.2 bicarbonate buffer and pH 13 sodium hydroxide buffer (FIG. 10). As anticipated, the substituted conjugates made from (3.60), (3.61), and (3.62) all underwent N-degradation readily in the pH 9.2 buffer. The faster N-degradation at lower pH supports the hypothesis that substitution on the guanidine nitrogens increases the cyclization kinetics.

[00564] As the N-conjugation reaction kinetics were significantly slower with substituted degradation agents, the kinetics of the degraders that did react with monomeric phenylalanine (FIG. 11) was measured. The unsubstituted degradation agent (3.45) approached 100% conversion between 30 and 40 minutes. This corroborates the earlier amine bead data observed with degradation agent (3.45), where full conversion was achieved within 45 minutes (FIG. 5). As shown in FIG. 12A, the formation of the products and completion of the reaction was confirmed by UPLC and MS. Additionally, the methyl and ethyl monosubstituted amidines (3.60) and (3.61) showed significantly slower kinetics, with apparent conversion around 40% after 1 hour. This apparent conversion was with a single amino acid, which exemplifies the further reduction in conversion with a peptide fragment that is significantly larger in comparison. The benzyl substituted degradation agent (3.62) showed the lowest conversion overall, which aligns with the angiotensin 1 data, as it converted only -10% overall after 16 hours. The UPLC and MS data for the methyl, ethyl, and benzyl analogues are provided in FIGs. 12B-12D

[00565] While the finding of increased N-degradation kinetics at lower pH through amidine nitrogen substitution proved fruitful, the significant decrease in N-terminal conjugation kinetics demonstrated that their reactivity toward N-terminal amines was severely hampered by the substitution. At times, there was no reaction with the peptide whatsoever. Due to the decrease in conjugation compared to the unsubstituted degradation agent, studies were continued with the unsubstituted degradation agent (3.45).

EXAMPLE 4. APPLICATION OF DEGRADATION AGENT FOR SOLUTION PHASE PEPTIDE DEGRADATION

[00566] Following the structure-activity relationship study of the photocaged amidines, next efforts were made to understand the scope of the degradation agent (3.45) by testing it with different peptide fragments. By testing degradation agent (3.45) with peptides containing various N-terminal amino acids, the robustness of the photocaged amidine as an N-terminal degrader was exemplified. As with angiotensin 1, a series of commercially available peptide fragments with different N-terminal amino acids was selected (FIG. 13).

[00567] In this assay, it is observed that conjugation to the N-terminal amine of each peptide was completed within 2 hours (FIG. 14). Interestingly, it is noted the conjugation of the lysine side chain y-amine for the EGRF fragment but not for the KRAS fragment. As the degrader was in excess during the reaction, it is assumed that all lysins may readily react with the degrader, forming an unreactive guanidine side chain. However, there appears to be a preference for the N-terminal over the lysine side chain y-amines, which makes sense considering the difference in pKa (N-terminus: 9-9.5 and lysine y-amine: 10.7). Additionally, when we acquired the calculated secondary structures of these fragments using AlphaFold, slight differences in their shapes were observed (FIG. 15).

[00568] In these calculated structures, the KRAS fragment forms a straight alpha helix, while the EGFR fragment contains a fold roughly halfway through the sequence. This fold in EGFR may reduce the exposure of the N-terminal amine and increase the exposure of the internal lysine for greater reactivity. KRAS, however, has a well-exposed N-terminal amine, and the degradation agent selectively reacted with the more reactive N-terminal amine when given the opportunity.

[00569] In terms of N-degradation, all tested and conjugated peptides underwent N- degradation in pH 13 buffer despite the changes to the N-terminal amino acid. This finding was promising in exemplifying the robustness of the degradation agent (3.45). While not all twenty amino acids were tested at the N-terminus, these selected peptide fragments represent a majority of the amino acid subtypes. Additionally, it was observed no degradation of the peptide conjugates in pH 13 buffer when left in the dark, demonstrating the full “trigger-and-release” capability of the photocaged degrader technology.

[00570] Finally, performing two N-terminal conjugations and degradations of the same peptide in solution was explored. To conduct this experiment, angiotensin 1 was reacted with degradation agent (3.45) and performed a photoinduced N-degradation. Unlike previous experiments, the degraded species was purified and reacted the degraded peptide, RVYIHPFHL (3.67), with (3.45) to perform another round of degradation, resulting in the twice-degraded species: RVYIHPFHL (3.68) (FIG. 16).

[00571] From this assay, it was observed iterative degradation of the peptide species, achieving full completion at each step. While the degraded peptide needed to be purified between the two degradation steps, this assay demonstrated that the novel degradation agent can be used iteratively for peptide sequencing. These two assays combined showcase the full potential application of a photocaged amidine sulfonate as an N-terminal peptide sequencing technology via N-degradation.

EXAMPLE 5. FLUOROSEQUENCING USING UNIVERSAL PROBES AND DEGRADATION AGENTS

[00572] A polypeptide is immobilized on a solid support for fluorosequencing. For example, a polypeptide may comprise the following residues: (N-terminus)-E-L-I-I-E-F-S-K-M-A-R-D-P- Q-R-Y-L-V-I-Q-G-N-E-R-M-(C -terminus) (SEQ ID NO: 1). The polypeptide is sequenced using a universal probe (2.108) of FIG. 19, and a degradation agent (3.45). To conduct fluoroesequecing, the polypeptide is first conjugated to a support to ensure single-molecule separation and spatial resolution.

Surface passivation and peptide conjugation

[00573] 384 well glass bottom plates with a #1.5H cover glass is first cleaned with 100 pL per well of 5 M NaOH solution. The solution is allowed to clean the glass surface for 1 h prior to washing 3x with 100 pL of ddH2O. Then 100 pL of Optima grade methanol is added and allowed to sit for an additional 30 min. Following this time, the methanol is removed and the surface is dried under nitrogen stream. Passivation and functionalization of the cleaned glass surface is performed by adding 100 pL of silanization mixture (Per 1 mL: 939 pL of Optima grade methanol, 50 pL of HPLC grade acetic acid, 10 pL of 1 M mPEG5-triethoxysilane DMSO, and 1 pL of 1 pM DBCO-PEG4-triethoxysilane) to wells. The multiwell plate is sealed using a 384 well silicon cap mat, and stirred on a shaker at 750 rpm at ambient temperature for 2 hours. Following this time, the silanization mixture is removed from the wells, and the wells are washed with 100 pL of Optima grade methanol 3x then dried under nitrogen stream.

[00574] Peptide conjugation and immobilization are performed by adding 100 pL of 1 pM peptide-azide solution in lx PBS to the functionalized well. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. The conjugated wells are then washed with 100 pL of lx PBS 5x.

[00575] Upon reaction, the C-terminal amino acid of the polypeptides is conjugated to the functionalized support, thereby immobilizing the polypeptides.

Probe conjugation on surface immobilized peptides

[00576] To the pre-conjugated peptide surface containing wells of a functionalized glass backed 384 multiwell plate, 100 pL of a 1 pM solution of probe (2.108) in pH 9.2 bicarbonate buffer is added. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. The probe-peptide conjugated wells are then washed with 100 pL of Optima grade methanol 3x then 100 pL of lx PBS 3x. Then 50 pL of the final imaging buffer (ImM Trolox in lx PBS) is then added to the wells.

[00577] This leads to conjugation of probes (2.108) to the N-terminal amino acid of the polypeptides. The probes (2.108) may recognize and bind to any amino acid positioned at the N- terminus of the polypeptide. Upon binding, the amino acid-probe conjugate exhibits a unique fluorescence profile (e.g., fluorescence intensity and fluorescence lifetime), that is characteristic to the specific amino acids that the probe is coupled to. This leads to binding of the probe to the N-terminal E residue (BOLDED) of the following polypeptide sequence: (N-terminus) Probe- E-L-I-I-E-F-S-K-M-A-R-D-P-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 1).

Surface measurements - Acquisition

[00578] Plates are imaged on a Leica Stellaris 8 Confocal Microscope with a 40x air immersion objective (0.95 NA). Regions of Interest (ROIs) within each well are chosen randomly using the Leica Navigator software feature, collecting 0.5 MP (512x512 pixels) with 25 nm step size to yield an approximate FOV size of 12.7 microns by 12.7 microns. Initial focus on the glass surface is determined first by XZ confocal scanning in reflection mode, then optimized in traditional XY mode to maximize contrast for observable spots in the field of view (FOV). Simultaneous spectral-lifetime imaging was proceeded with excitation at 440 nm with 5% laser power and a 20 MHz pulse repetition rate. Spectral acquisition is ranged from 450 nm- 640 nm in 5 nm steps with 10 nm bin width for a total of 39 steps. Confocal pinhole diameter is set to 2 AU using 550 nm estimation. Line scan rate is 200 Hz for a pixel dwell time of approximately 7.7 microseconds per pixel.

[00579] The acquired fluorescence images are then analyzed. Fluorescence intensities and fluorescence lifetime are extracted from region of interest corresponding to individual polypeptide spots. Emission profiles and fluorescence lifetime are analyzed against a reference database to identify the presence and identify of the labeled amino acid at each sequencing position. Upon comparing to the reference database, identity of the N-terminal amino acid (e.g., “E”) is identified.

Photoinduced Degradation of the Probe

[00580] After data acquisition, the probe (2.108) is subjected to photoinduced degradation. Upon irradiation of the wells comprising probe-amino acid (of the immobilized polypeptide) conjugates with 365 nm LEDs over two hours in the presence of oxygen, a clear decrease in absorbance at 425 nm is observed, indicating that the probe is detached from the amino acid (of the immobilized polypeptide). Upon photoinduced degradation, the well is washed to remove any residual probes. The photoinduced degradation of probe leaves the original amino acid of the polypeptide: (N-terminus) E-L-I-I-E-F-S-K-M-A-R-D-P-Q-R-Y-L-V-LQ-G-N-E-R-M-(C- terminus) (SEQ ID NO: 1).

Degrader conjugation and N-degradation on surface immobilized peptides

[00581] To the immobilized polypeptide, 100 pL of a 1 pM solution of a degradation agent (3.45) is added in pH 9.2 bicarbonate buffer. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. Following this time, the wells are washed with 100 pL of lx PBS 5x.

[00582] This leads the degradation agent to be coupled to the polypeptide: (N-terminus) Degradation Agent-E-L-I-I-E-F-S-K-M-A-R-D-P-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 1).

[00583] The degradation agent -polypeptide conjugate is then taken up in a pH 10 bicarbonate buffer, and irradiated with 10 W 365 nm LEDs for 5 minutes. Following this time, the solution is allowed to shake on a shaker at 750 rpm at ambient temperature for 5 hours. Following this time, the well is washed with 100 pL of lx PBS 5x.

[00584] Following the degradation step, the N-terminal amino acid of the immobilized polypeptide is cleaved and removed, thereby exposing the next amino acid residue at the N- terminus. This reaction results in removal of the N-terminal amino acid: (N-terminus) L-I-I-E-F- S-K-M-A-R-D-P-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 2).

[00585] This newly exposed N-terminal residue is then available for additional cycles (e.g., Cycle 2). To identify the next amino acid (the newly exposed N-terminal residue), the polypeptide is conjugated to the probe (2.108). This allows the probeto be conjugation to the newly exposed N-terminal amino acid: (N-terminus) Probe-L-I-I-E-F-S-K-M-A-R-D-P-Q-R-Y- L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 2).

[00586] Upon conjugation, emission profile (e.g., emission intensity or lifetime) is detected. The identity of the terminal amino acid is determined (e.g., “L”) by comparing the emission profile to the reference. Upon fluorescence detection, the probe is de-coupled (cleaved) from the N-terminal amino acid upon exposing to light as described above: (N-terminus)-L-I-I-E-F-S-K- M-A-R-D-P-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 2). The polypeptide is then contacted with a degradation agent described herein: (N-terminus)-Degradation Agent-L- I-I-E-F-S-K-M-A-R-D-P-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 2).

Degradation agent, upon exposure to light, cleaves the N-terminal amino acid, leaving the next newly exposed N-terminal residue: : (N-terminus)-I-I-E-F-S-K-M-A-R-D-P-Q-R-Y-L-V-I-Q-G- N-E-R-M-(C -terminus) (SEQ ID NO: 3). The cycles may be repeated until all or a subset of amino acid residues of the polypeptide is identified.

EXAMPLE 6. FLUOROSEQUENCING USING AMINO ACID SPECIFIC PROBES AND DEGRADATION AGENTS

[00587] A polypeptide is immobilized on a solid support for fluorosequencing. The polypeptide is sequenced using amino acid specific probes and a degradation agent (3.45). [00588] The polypeptide of interest is prepared from a biological sample, and immobilized onto a functionalized glass coverslip via click chemistry. The polypeptide is first fluorescently labeled using a panel of amino acid-specific probes, each conjugated to a spectrally distinct fluorophore. Lysine residues are labeled with Alex Fluor 488 (Bolded), tyrosine residues are labeled with Alexa Fluor 555 (Underlined), and cysteine residues are labeled with Alexa Fluor 647 (Italicized), using chemoselective reagents designed to bind to side chains. The specific amino acids of the polypeptide are labeled with the one or more probes described herein: (N- terminus) E-L-I-I-E-F-S-K-M-A-R-D-P-C-C-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 4).

[00589] After labeling, baseline fluorescence images (cycle 0) are acquired using a fluorescence microscope. The sequencing procedure is then initiated through a cycle of N- terminal degradation via a degradation agent (3.45) by conjugating the degradation agent to the N-terminal amino acid and, subsequently, degrading the N-terminal amino acid upon exposure of the polypeptide to light (e.g., with 365 nm LEDs for 5 minutes).

[00590] The degradation agent (3.45) is added in pH 9.2 bicarbonate buffer for conjugation to the polypeptide. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. Following this time, the polypeptide is washed with 100 pL of lx PBS 5x. This leads to conjugation of the degradation agent to the polypeptide: (N-terminus) Degradation Agent-E-L-I-I-E-F-S-K-M-A-R-D-P-C-C-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C- terminus) (SEQ ID NO: 4).

[00591] The degrader-polypeptide conjugate is then taken up in pH 10 bicarbonate buffer, and irradiated with 10 W 365 nm LEDs for 5 minutes. Following this time, the solution is allowed to shake on a shaker at 750 rpm at ambient temperature for 5 hours. Following this time, the polypeptide is washed with 100 pL of lx PBS 5x. The exposure of the polypeptide to light leads the N-terminal amino acid of the polypeptide to be degraded: (N-terminus) L-I-I-E-F-S-K- M-A-R-D-P-C-C-Q-R-Y-L-V-LQ-G-N-E-R-M-(C-terminus) (SEQ ID NO: 5). Then, fluorescence images (cycle 1) are acquired using the fluorescence microscope.

[00592] This N-terminal degradation exposes the next residue in sequence. Then, the cycle (e.g., N-terminal degradation and detection of the signal) is repeated. The polypeptide (e.g., having one fewer amino acid) is then conjugated to the N-terminal amino acids: (N-terminus) Degradation Agent-L-I-I-E-F-S-K-M-A-R-D-P-C-C-Q-R-Y-L-V-I-Q-G-N-E-R-M-(C-terminus) (SEQ ID NO: 5). The exposure of polypeptide to light leads the next N-terminal amino acid to be degraded: (N-terminus) I-I-E-F-S-K-M-A-R-D-P-C-C-Q-R-Y-L-V-LQ-G-N-E-R-M-(C- terminus) (SEQ ID NO: 6). Then, fluorescence images (cycle 2) are acquired using the fluorescence microscope.

[00593] This process - N-terminal cleavage followed by imaging- is repeated over 20-30 cycles to progressively reveal the sequence of each polypeptide.

[00594] The fluorescence signals or signal change in each polypeptide spot are tracked across cycle. A sharp decrease in fluorescence intensity in a specific channel at a specific cycle is interpreted as the removal of a labeled amino acid at that sequence position. Image analysis software is used to align the images, segment individual peptide spots, and extract intensity trances. The fluorescence decay profiles are interpreted by a base-calling algorithm that assigns amino acid identity to each position in the peptide, based on probe identity and signal drop cycle. These reads are aligned to a reference proteome or used for de novo sequencing application.

EXAMPLE 7. FLUOROSEQUENCING USING UNIVERSAL PROBES AND DEGRADATION AGENTS OF A PLURALITY SAMPLES

[00595] A sample comprising a population of polypeptides immobilized on a solid support is subjected to fluorosequencing. The population of peptides is sequenced using a universal probe (2.108) of FIG. 19, and a degradation agent (3.45). To conduct fluoroesequecing, each polypeptide of the population of polypeptides is first conjugated to a support to ensure singlemolecule separation and spatial resolution.

Surface passivation and peptide conjugation

[00596] 384 well glass bottom plates with a #1.5H cover glass is first cleaned with 100 pL per well of 5 M NaOH solution. The solution is allowed to clean the glass surface for 1 h prior to washing 3x with 100 pL of ddH2O. Then 100 pL of Optima grade methanol is added and allowed to sit for an additional 30 min. Following this time, the methanol is removed and the surface is dried under nitrogen stream. Passivation and functionalization of the cleaned glass surface is performed by adding 100 pL of silanization mixture (Per 1 mL: 939 pL of Optima grade methanol, 50 pL of HPLC grade acetic acid, 10 pL of 1 M mPEG5-triethoxysilane DMSO, and 1 pL of 1 pM DBCO-PEG4-triethoxysilane) to wells. The multiwell plate is sealed using a 384 well silicon cap mat, and stirred on a shaker at 750 rpm at ambient temperature for 2 hours. Following this time, the silanization mixture is removed from the wells, and the wells are washed with 100 pL of Optima grade methanol 3x then dried under nitrogen stream.

[00597] Peptide conjugation and immobilization are performed by adding 100 pL of 1 pM peptide-azide solution in lx PBS to the functionalized well. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. The conjugated wells are then washed with 100 pL of lx PBS 5x.

[00598] Upon reaction, each C-terminal amino acid of the population of polypeptides is conjugated to the functionalized support, thereby immobilizing the population of polypeptides.

Probe conjugation on surface immobilized peptides

[00599] To the pre-conjugated peptide surface containing wells of a functionalized glass backed 384 multiwell plate, 100 pL of a 1 pM solution of probe (2.108) in pH 9.2 bicarbonate buffer is added. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. The probe-peptide conjugated wells are then washed with 100 pL of Optima grade methanol 3x then 100 pL of lx PBS 3x. Then 50 pL of the final imaging buffer (ImM Trolox in lx PBS) is then added to the wells.

[00600] This leads to conjugation of probes (2.108) to all N-terminal amino acids of the population of polypeptides. The probes (2.108) may recognize and bind to any amino acid positioned at N-terminus of the polypeptide. Upon binding, each amino acid-probe conjugate exhibits a unique fluorescence profile (e.g., fluorescence intensity and fluorescence lifetime), that is characteristic to the specific amino acids.

Surface measurements - Acquisition

[00601] Plates are imaged on a Leica Stellaris 8 Confocal Microscope with a 40x air immersion objective (0.95 NA). Regions of Interest (ROIs) within each well are chosen randomly using the Leica Navigator software feature, collecting 0.5 MP (512x512 pixels) with 25 nm step size to yield an approximate FOV size of 12.7 microns by 12.7 microns. Initial focus on the glass surface is determined first by XZ confocal scanning in reflection mode, then optimized in traditional XY mode to maximize contrast for observable spots in the field of view (FOV). Simultaneous spectral -lifetime imaging was proceeded with excitation at 440 nm with 5% laser power and a 20 MHz pulse repetition rate. Spectral acquisition is ranged from 450 nm- 640 nm in 5 nm steps with 10 nm bin width for a total of 39 steps. Confocal pinhole diameter is set to 2 AU using 550 nm estimation. Line scan rate is 200 Hz for a pixel dwell time of approximately 7.7 microseconds per pixel.

[00602] The acquired fluorescence images are then analyzed. Fluorescence intensities and fluorescence lifetime are extracted from region of interest corresponding to individual polypeptide spots. Emission profiles and fluorescence lifetime are analyzed against a reference database to identify the presence and identify of the labeled amino acid at each sequencing position.

Photoinduced Degradation of the Probe

[00603] After data acquisition, the probe (2.108) is subjected to photoinduced degradation. Upon irradiation of the wells comprising probe-amino acid (of the immobilized polypeptide) conjugates with 365 nm LEDs over two hours in the presence of oxygen, a clear decrease in absorbance at 425 nm is observed, indicating that the probe is detached from the amino acid (of the immobilized polypeptide). Upon photoinduced degradation, the wells are washed to remove any residual probes. Degrader conjugation and N-degradation on surface immobilized peptides

[00604] To the immobilized polypeptide, 100 pL of a 1 pM solution of a degradation agent (3.45) is added in pH 9.2 bicarbonate buffer. The solution is allowed to shake under darkness on a shaker at 750 rpm at ambient temperature for 1 hour. Following this time, the wells are washed with 100 pL of lx PBS 5x.

[00605] The degrader-peptide conjugate wells are then taken up in a pH 10 bicarbonate buffer, and irradiated with 10 W 365 nm LEDs for 5 minutes. Following this time, the solution is allowed to shake on a shaker at 750 rpm at ambient temperature for 5 hours. Following this time, the wells are washed with 100 pL of lx PBS 5x.

[00606] Following the degradation step, the N-terminal amino acid of the immobilized polypeptide is cleaved and removed, thereby exposing the next amino acid residue at the N- terminus.

[00607] This newly exposed N-terminal residue is then available for additional cycles of probe (2.108) conjugation, fluorescence detection, and degradation to identify the amino acid sequence of each polypeptide.

EXAMPLE 8. FLUOROSEQUENCING USING AMINO ACID SPECIFIC PROBES AND DEGRADATION AGENTS A PLURALITY SAMPLES

[00608] A sample comprising a population of polypeptides immobilized on a solid support is subjected to fluorosequencing. The population of peptides is sequenced using amino acid specific probes and a degradation agent (3.45).

[00609] A sample containing polypeptide of interest is prepared from a biological sample, and immobilized onto a functionalized glass coverslip via click chemistry. The polypeptides are fluorescently labeled using a panel of amino acid-specific probes, each conjugated to a spectrally distinct fluorophore. Lysine residues are labeled with Alex Fluor 488, tyrosine residues are labeled with Alexa Fluor 555, and cysteine residues are labeled with Alexa Fluor 647, using chemoselective reagents designed to bind to side chains.

[00610] After labeling, baseline fluorescence images (cycle 0) are acquired using a fluorescence microscope. The sequencing procedure is then initiated through a cycle of N- terminal degradation via a degradation agent (3.45). This N-terminal degradation exposes the next residue in sequence. The surface is then washed and re-images in all fluorescence channels (Cycle 1). This process - N-terminal cleavage followed by imaging- is repeated over 20-30 cycles to progressively reveal the sequence of each polypeptide. [00611] The fluorescence signals in each polypeptide spot are tracked across cycle. A sharp decrease in fluorescence intensity in a specific channel at a specific cycle is interpreted as the removal of a labeled amino acid at that sequence position. Image analysis software is used to align the images, segment individual peptide spots, and extract intensity trances. The fluorescence decay profiles are interpreted by a base-calling algorithm that assigns amino acid identity to each position in the peptide, based on probe identity and signal drop cycle. These reads are aligned to a reference proteome or used for de novo sequencing application.

[00612] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method, comprising:

(a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide, comprising a photo-cleavable moiety; and

(b) subjecting at least a portion of the first modified polypeptide to a condition sufficient to generate a second modified polypeptide and a fragment comprising a residue of the terminus of the polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

2. The method of claim 1, wherein the photo-cleavable moiety is cleavable when subjected to light comprising a wavelength from 200 nm to 750 nm.

3. The method of claim 1 or 2, wherein the condition sufficient to generate the second modified polypeptide comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or a combination thereof.

4. The method of claim 3, wherein the buffer condition comprises an aqueous basic buffer.

5. The method of claim 3, wherein the condition sufficient to generate the second modified polypeptide comprises the light source and/or the aqueous basic buffer.

6. The method of claim 5, wherein the light source comprises a wavelength from about 200 nm to about 750 nm.

7. The method of claim 5 or 6, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0.

8. A method, comprising:

(a) contacting a terminus of a polypeptide with a degradation agent, thereby forming a first modified polypeptide; and

(b) subjecting at least a portion of the first modified polypeptide to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the second modified polypeptide comprises one or more fewer amino acids than the first polypeptide.

9. The method of claim 8, wherein (b) comprises generating a fragment comprising a residue of the terminus of the polypeptide.

10. The method of claim 8 or 9, wherein the one or more wavelengths is from about 200 nm to about 750 nm.

11. The method of any one of claims 8-10, wherein (b) comprises subjecting the first modified polypeptide to one or more additional conditions sufficient to generate the second modified polypeptide.

12. The method of claim 11, wherein the one or more additional conditions comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent, or a combination thereof.

13. The method of claim 12, wherein the buffer condition comprises an aqueous basic buffer.

14. The method of claim 11 or 12, wherein the one or more additional conditions comprises an aqueous basic buffer.

15. The method of claim 14, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0.

16. The method of claim 12, wherein the temperature comprises a reaction temperature.

17. The method of claim 16, wherein the one or more additional conditions comprises a reaction temperature from about 30 degrees to 60 degrees.

18. The method of any one of the preceding claims, wherein the degradation agent comprises a photo-cleavable moiety.

19. The method of any one of the preceding claims, wherein the polypeptide is coupled to a biomolecule.

20. The method of any one of the preceding claims, wherein the degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety.

21. The method of any one of the preceding claims, further comprising, prior to (a), providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes.

22. The method of claim 21, wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids.

23. The method of claim 21, wherein a probe of the one or more probes is specific to an amino acid type.

24. The method of claim 22 or 23, wherein the probe is covalently coupled to the polypeptide.

25. The method of any one of claims 22-24, wherein the probe is coupled to hydroxyl, a carboxylic, an amino, a thiol group of the amino acid of the polypeptide or any combination thereof.

26. The method of any one of claims 21-25, further comprising detecting one or more signals or signal change from the one or more probes to identify a characteristic of at least a portion of the polypeptide.

27. The method of claim 26, wherein the one or more signals or signal change comprises one or more fluorescent spectral properties.

28. The method of claim 26 or 27, further comprising using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide.

29. The method of any one of the preceding claims, wherein the polypeptide is coupled to a surface or support.

30. The method of any one of the preceding claims, further comprising repeating (a) and (b) one or more times to degrade one or more subsequent terminal amino acids of the polypeptide.

31. The method of any one of the preceding claims, wherein the polypeptide is among a sample comprising a plurality of analytes.

32. The method of any one of the preceding claims, further comprising identifying a terminal amino acid of the polypeptide by comparing one or more spectral properties generated by the probe to a plurality of reference spectral properties.

33. A method for sample analysis, comprising:

(a) providing a sample comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is coupled to a first one or more probes and the second polypeptide is coupled to a second one or more probes;

(b) detecting (1) one or more signals or signal change from the first one or more probes and (2) one or more signal or signal change from the second one or more probes;

(c) contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent, thereby removing at least one amino acid from the first polypeptide and/or from the second polypeptide, wherein the first degradation agent comprises a first photo-cleavable moiety and/or the second degradation agent comprises a second photo-cleavable moiety; and

(d) identifying one or more characteristics of the sample.

34. The method of claim 33, repeating (a) and (b) one or more additional times on one or more subsequent amino acids of the first polypeptide and/or the second polypeptide to generate one or more first additional modified polypeptides and/or one or more second additional modified polypeptides, respectively.

35. The method of claim 33 or 34, wherein the one or more characteristics of the sample comprises a number of polypeptides in the sample, type of polypeptides in the sample, an origin of the sample, impurities in the sample, presence of a polypeptide, absence of a polypeptide, or any combination thereof.

36. The method of any one of claims 33-35, wherein the first degradation agent and/or the second degradation agent comprises a photo-cleavable moiety.

37. The method of any one of claims 33-36, wherein the first degradation agent and/or the second degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

38. The method of any one of claims 33-37 wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises one or more aromatic groups.

39. The method of any one of claims 33-37, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises a nitro-substituted benzyl group.

40. The method of any one of claims 33-39, further comprising repeating (a), (b), and (c) one or more times to detect one or more subsequent terminal amino acids of the first polypeptide and/or the second polypeptide.

41. The method of any one of claims 33-40, wherein the first polypeptide is coupled to a first biomolecule and/or the second polypeptide is coupled to a second biomolecule.

42. The method of any one of claims 33-41, wherein (1) a first probe of the first one or more probes is coupled to a terminal amino acid of the first polypeptide and/or (2) a second probe of the second one or more probes is coupled to a terminal amino acid of the second polypeptide.

43. The method of any one of claims 33-42, wherein a first probe of the first one or more probes and/or a second probe of the second one or more probes exhibits different spectral properties when conjugated to different amino acids.

44. The method of any one of claims 33-43, wherein a first probe of the first one or more probes and/or a second probe of the second one or more probes is specific to an amino acid type.

45. The method of any one of claims 33-44, wherein (b) and/or (c) comprises determining (1) a first property of at least a portion of the first polypeptide and/or (2) a second property of at least a portion of the second polypeptide.

46. The method of any one of claims 33-45, wherein (1) the one or more signals or signal change from the first one or more probes and/or (2) the one or more signal or signal change from the second one or more probes comprises one or more fluorescent spectral properties.

47. The method of any one of claims 33-46, further comprising using at least i) the one or more signals or signal change from the first one or more probes and/or the one or more signal or signal change from the second one or more probes, and/or ii) removed at least one amino acid from the first polypeptide and/or from the second polypeptide to identify the one or more characteristics of the at least the portion of the first polypeptide and/or the at least the portion of the second polypeptide.

48. The method of any one of claims 33-47, wherein the first polypeptide and/or the second polypeptide is coupled to a surface or support.

49. The method of any one of claims 33-48, wherein the sample is a biological sample.

50. The method of any one of claims 33-49, wherein the first degradation agent and/or the second degradation agent comprises the same chemical structure.

51. The method of any one of claims 34-50, wherein (c) comprises subjecting the first polypeptide and the second polypeptide to one or more conditions sufficient to generate the one or more first additional modified polypeptides and/or the one or more second additional modified polypeptides.

52. The method of claim 51, wherein the one or more conditions sufficient to generate the one or more first additional modified polypeptides and/or the one or more second additional modified polypeptides comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of a degradation agent, or any combination thereof.

53. The method of claim 51, wherein the one or more conditions comprises a light source and/or an aqueous basic buffer.

54. The method of claim 53, wherein the aqueous basic buffer comprises a pH value from about 8.0 to about 13.0.

55. The method of any one of claims 33-54, further comprising identifying a terminal amino acid of the first polypeptide and/or the second polypeptide by comparing spectral properties of the first one or more probes and/or the second one or more probes to a plurality of reference spectral properties.

56. A compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and

PC is a photo-cleavable moiety.

57. The compound of claim 56, wherein the photo-cleavable moiety comprises one or more aromatic groups.

58. The compound of claim 56 or 57, wherein the photo-cleavable moiety is cleavable when subjected to a light source having a wavelength of about 200 to about 750 nm.

59. The compound of any one of claims 56-58, wherein the photo-cleavable moiety comprises a nitro- substituted benzyl group.

60. The compound of any one of claims 56-59, wherein the degradation agent is

61. A method, compri sing :

(a) providing a polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes;

(b) detecting one or more signals or signal change from the one or more probes; and

(c) subjecting at least a portion of the polypeptide to (1) a first light comprising a first one or more wavelengths sufficient to remove the one or more probes, and (2) second light comprising a second one or more wavelengths sufficient to remove a terminal amino acid of the polypeptide.

62. The method of claim 61, repeating (a) and (b) one or more additional times on one or more subsequent amino acids of the polypeptide to generate one or more modified polypeptides.

63. The method of claim 61 or 62, wherein (c) comprises contacting a terminus of the polypeptide with a degradation agent.

64. The method of claim 63, wherein the degradation agent comprises a photo-cleavable moiety.

65. The method of any one of claims 61-64, wherein the first one or more wavelength and/or the second one or more wavelengths is from 200 nm to 750 nm.

66. The method of any one of claims 61-65, wherein a probe of the one or more probes is removed prior to removal of the terminal amino acid of the polypeptide.

67. The method of any one of claims 61-65, wherein a probe of the one or more probes is removed subsequent to removal of the terminal amino acid of the polypeptide.

68. The method of any one of claims 61-67, further comprising determining at least one characteristic of the at least the portion of the polypeptide.

69. The method of any one of claims 61-68, wherein the detecting of (b) occurs prior to (c).

70. The method of any one of claims 61-68, wherein the detecting of (b) occurs subsequent to (c).

71. The method of any one of claims 64-70, wherein the photo-cleavable moiety comprises one or more aromatic groups.

72. The method of any one of claims 64-70, wherein the photo-cleavable moiety comprises a nitro-substituted benzyl group.

73. The method of any one of claims 61-72, wherein the polypeptide is coupled to a surface or support.

74. The method of any one of claims 62-73, wherein (c) comprises subjecting the at least a portion of the polypeptide to one or more conditions sufficient to generate the one or more modified polypeptides.

75. The method of claim 74, wherein the one or more conditions sufficient to generate the one or more modified polypeptide comprises a pH condition, an optical condition, a duration condition, a buffer condition, temperature, partition coefficient value, water solubility of the degradation agent or any combination thereof.

76. A method, comprising:

(b) subjecting at least a portion of the first modified polypeptide in a solution to light comprising one or more wavelengths sufficient to generate a second modified polypeptide, wherein the solution comprises one or more acids, and wherein the second modified polypeptide comprises one or more fewer amino acids than the polypeptide.

77. The method of claim 76, wherein the degradation agent comprises a photo-cleavable moiety.

78. The method of claim 76 or 77, wherein the one or more acids comprises a Lewis acid.

79. The method of any one of claims 76-78, wherein pH of the solution is from 7 to 13.

80. The method of any one of claims 76-79, wherein the solution comprises the one or more acids at a concentration from 0.1 M to 1 M.

81. The method of any one of claims 76-80, wherein the light comprises a wavelength from 200 nm to 750 nm.

82. The method of any one of claims 77-81, wherein the photo-cleavable moiety comprises one or more aromatic groups.

83. The method of any one of claims 77-81, wherein the photo-cleavable moiety comprises a nitro-substituted benzyl group.

84. The method of any one of claims 76-83, wherein, prior to (a), the method further comprises providing the polypeptide, wherein the polypeptide comprises one or more amino acids coupled to one or more probes; and wherein a probe of the one or more probes exhibits different spectral properties when conjugated to different amino acids.

85. The method of claim 84, further comprising detecting one or more signals or signal change from the one or more probes to identify a characteristic of at least a portion of the polypeptide.

86. The method of claim 85, further comprising using at least i) the one or more signals or signal change and/or ii) the fragment comprising the residue to identify the characteristic of the at least the portion of the polypeptide.

87. The method of any one of claims 76-86, wherein the polypeptide is coupled to a surface or support.

88. The method of any one of claims 76-87, wherein the polypeptide is among a sample comprising a plurality of analytes.

89. The method of any one of claims 76-88, further comprising identifying a terminal amino acid of the polypeptide by comparing spectral properties of the probe to a plurality of reference spectral properties.

90. A method for analyzing a sample, comprising:

(a) providing a first polypeptide to a first location on a first support and a second polypeptide to a second location on a second support, wherein the first polypeptide is coupled to first one or more probes;

(b) detecting one or more signals or signal change from the one or more probes of the first polypeptide; and

(c) selectively subjecting at least a portion of the first polypeptide to light comprising one or more wavelengths sufficient to selectively remove a terminal amino acid of the first polypeptide.

91. The method of claim 90, repeating (b) and (c) one or more additional times on one or more subsequent amino acids of the first polypeptide to generate one or more additional modified polypeptide.

92. The method of claim 90 or 91, wherein (c) comprises contacting a terminus of the first polypeptide with a first degradation agent and/or a terminus of the second polypeptide with a second degradation agent.

93. The method of claim 92, wherein the first degradation agent and/or the second degradation agent comprises a photo-cleavable moiety.

94. The method of claim 92 or 93, wherein (c) comprises subjecting the at least the portion of the first polypeptide to the first degradation agent to selectively remove the terminal amino acid of the first polypeptide.

95. The method of any one of claims 90-94, wherein (c) comprises providing the light to the at least the portion of the first polypeptide at the first location of the first support.

96. The method of any one of claims 90-95, wherein (c) comprises providing the light to the at least the portion of the first polypeptide at the first location of the first support, but not to at least a portion of the second polypeptide at the second location of the second support.

97. The method of any one of claims 90-96, wherein the selectively subjecting comprises (1) subjecting the at least the portion of the first polypeptide to the light at a first time and (2) subjecting at least a portion of the second polypeptide to another light at a second time.

98. The method of claim 97, wherein the second time is subsequent to the first time.

99. The method of any one of claims 90-98, wherein the first support is the same support as the second support.

100. The method of any one of claims 90-99, wherein a distance between the first location and the second location is at least about 150 nm.

101. The method of any one of claims 92-100, wherein the first degradation agent and/or the second degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group;

PC is a photo-cleavable moiety.

102. The method of any one of claims 92-101, wherein the first degradation agent and/or the second degradation agent comprises a first photo-cleavable moiety and/or a second photo- cleavable moiety, respectively.

103. The method of claim 102, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises one or more aromatic groups.

104. The method of claim 102, wherein the first photo-cleavable moiety and/or the second photo-cleavable moiety comprises a nitro- substituted benzyl group.

105. The method of any one of claims 90-104, wherein the sample is in an aqueous basic buffer.

106. The method of claim 105, wherein the aqueous basic buffer comprises a pH value from about 9.0 to about 13.0.

107. A method, comprising:

(a) detecting one or more signals or signal change from one or more probes coupled to a polypeptide;

(b) subjecting at least a portion of the polypeptide to a light condition sufficient to remove a terminal amino acid of the polypeptide; and

(c) using the one or more signal or signal change to determine one or more characteristics of the at least the portion of an analyte with an accuracy of at least 60%.

108. The method of claim 107, wherein the one or more probes is coupled to one or more amino acids of the polypeptide.

109. The method of claim 107 or 108, wherein the one or more signals or signal change determines the one or more characteristics of the at least the portion of the analyte with an accuracy of at least 85%.

110. The method of any one of claims 107-109, further comprising, prior to (b), contacting a terminus of the polypeptide with a degradation agent.

111. The method of claim 110, wherein the degradation agent comprises a photo-cleavable moiety.

112. The method of claim 110, wherein the degradation agent is a compound of Formula I, or a salt, solvate, or a derivative thereof wherein:

LG is a leaving group; R¹ and R² are independently hydrogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, or 3- to 10-membered heterocyclyl, wherein the alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl is optionally substituted; and PC is a photo-cleavable moiety.