WO2023019148A1 - Macromolecule enrichment - Google Patents

Macromolecule enrichment Download PDF

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Publication number
WO2023019148A1
WO2023019148A1 PCT/US2022/074724 US2022074724W WO2023019148A1 WO 2023019148 A1 WO2023019148 A1 WO 2023019148A1 US 2022074724 W US2022074724 W US 2022074724W WO 2023019148 A1 WO2023019148 A1 WO 2023019148A1
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WIPO (PCT)
Prior art keywords
moiety
multifunctional molecule
group
molecule
macromolecule
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PCT/US2022/074724
Other languages
French (fr)
Inventor
Preston B. WILLIAMS
Bruce Wilcox
Chinmay BELTHANGADY
Philip Ma
Jared DEYARMIN
Manoj KHADKA
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PrognomIQ, Inc.
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Publication of WO2023019148A1 publication Critical patent/WO2023019148A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the delivery moiety comprises an alkyne. In some aspects, the alkyne comprises a terminal alkyne. In some aspects, the alkyne is a cyclic alkyne. In some aspects, the cyclic alkyne is a DBCO moiety, azacyclooctyne moiety, or cyclooctyne moiety. In some aspects, the cyclic alkyne is a DBCO moiety. In some aspects, the delivery moiety comprises an azide. In some aspects, the reactive moiety comprises a hydrazide.
  • the reactive moiety comprises -C(O)NHNHC(O)OtBu, - C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2. In some aspects, the reactive moiety comprises - C(O)NHNHC(O)OtBu or -C(O)NHNH2. In some aspects, the reactive moiety comprises - C(O)NHNHC(O)OtBu. In some aspects, the reactive moiety comprises -C(O)NHNH2. In some aspects, R 3 comprises the enrichment handle, and wherein the enrichment handle comprises biotin, desbiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof.
  • the enrichment handle comprises biotin or desbiotin. In some aspects, the enrichment handle comprises biotin, wherein the cleavable moiety is cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof.
  • the cleavable moiety is cleaved by light, an enzyme, or a combination thereof.
  • the light comprises UV light, visible light, IR light, laser, or a combination thereof.
  • the cleavable moiety comprises a photocleavable moiety.
  • the photocleavable moiety comprises a o-nitrobenzyloxy group, o- nitrobenzyl amino group, o-nitrobenzyl group, o-nitroveratryl group, phenacyl group, p- alkoxyphenacyl group, benzoin group, pivaloyl group, or a benzyl halide.
  • the photocleavable moiety comprises the o-nitrobenzyl group.
  • the o-nitrobenzyl group is substituted with a methoxy group or an ethoxy group.
  • the photocleavable moiety is represented by the formula:
  • n is selected from any number from 0 to 10. In some aspects, n is 3. In some aspects, the benzyl halide comprises -F, -Cl, -I, -Br, or a combination thereof. In some aspects, each of the connecting groups of X 1 , Y 1 , X 2 , Y 2 , X 3 , and Y 3 is independently selected from a group consisting of: a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate, and
  • each of the connecting groups of X 1 , Y 1 , X 2 , Y 2 , X 3 , and Y 3 is independently selected from a group consisting of: an ester, an amine, an amide, a carbamate, a urea, a thiourea, a triazole, and a carbonate.
  • the heteroalkylene of L la , L 2a , L 3a , L lb , L 2b , or L 3b is a PEG1-20 or a derivative thereof.
  • the heteroalkylene of L la , L 2a , L 3a , L lb , L 2b , or L 3b is a PEG4 or a derivative thereof.
  • the alkylene of L la , L 2a , L 3a , L lb , L 2b , or L 3b is a C1-C10 alkylene or a derivative thereof.
  • a multifunctional molecule comprising: a central moiety, a first arm comprising a delivery moiety connected to the central moiety, and a second arm comprising a reactive group connected to the central moiety; contacting a sample comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule.
  • the method enriches the macromolecule for further assessment.
  • the central moiety comprises a tertiary amine.
  • the delivery moiety is activated with a delivery agent that binds the macromolecule.
  • the delivery agent is azide-modified.
  • the delivery moiety comprises an alkyne. In some aspects, the delivery moiety comprises DBCO moiety, azacyclooctyne moiety or cyclooctyne moiety. In some aspects, the delivery moiety comprises a dibenzocyclooctyne (DBCO) moiety.
  • the first arm comprises a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety. In some aspects, the PEG linker of the first arm comprises PEG1-20. In some aspects, the PEG linker of the first arm comprises PEG2-8. In some aspects, the PEG linker of the first arm comprises PEG4.
  • the second arm comprises a PEG linker connecting the reactive group to the central moiety.
  • the PEG linker of the second arm comprises PEG1-20.
  • the PEG linker of the second arm comprises PEG2-8.
  • the PEG linker of the second arm comprises PEG4.
  • the reactive group comprises a hydrazide reactive group.
  • the reactive group comprises -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2.
  • the reactive group comprises -C(O)NHNH2.
  • the multifunctional molecule further comprises a third arm comprising an enrichment handle connected to the central moiety.
  • the third arm comprises a hydrocarbon connecting the enrichment handle to the central moiety.
  • the enrichment handle comprises biotin or desthiobiotin.
  • the enrichment handle comprises biotin.
  • the multifunctional molecule comprises N-(DBCO-PEGn)-N-Biotin-PEG m -hydrazide or a salt thereof.
  • n comprises 1-20.
  • n comprises 2-8.
  • m comprises 1-20.
  • m comprises 2-8.
  • the multifunctional molecule comprises some aspects, the multifunctional molecule comprises some aspects, the multifunctional molecule comprises some aspects, the multifunctional molecule comprises
  • the multifunctional molecule is provided in solution. In some aspects, the multifunctional molecule is provided as a salt with a counterion. In some aspects, the counterion comprises trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • Some aspects include solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule. Some aspects include activating copper-free click chemistry between the delivery moiety and an azide-modified delivery agent prior to contacting the sample with the multifunctional molecule. Some aspects include activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule. In some aspects, activating the click chemistry includes mixing.
  • Some aspects include oxidizing the sample, thereby oxidizing the macromolecule, prior to contacting the sample with the multifunctional molecule. In some aspects, oxidizing the macromolecule generates an aldehyde of the macromolecule, and wherein the aldehyde reacts with the reactive group. Some aspects include binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule. In some aspects, the affinity reagent is connected to a solid support. In some aspects, the affinity reagent comprises avidin or streptavidin. Some aspects include concentrating the captured multifunctional molecule bound to the macromolecule. In some aspects, concentrating comprises precipitating, centrifuging, eluting, or a combination thereof.
  • Some aspects include releasing at least part of the macromolecule from the multifunctional molecule. Some aspects include assaying the macromolecule. In some aspects, the assaying comprises performing mass spectrometry. Some aspects include identifying the macromolecule in the sample as indicative of a biological state. In some aspects, the biological state comprises a disease state. Some aspects include identifying the subject as having the biological state. In some aspects, the macromolecule comprises a protein, carbohydrate, lipid, metabolite or nucleic acid. In some aspects, the protein comprises a post-translational modification. In some aspects, the post- translational modification comprises glycosylation. In some aspects, the protein comprises a glycoprotein or proteoglycan. In some aspects, the sample comprises a biological sample.
  • the biological sample comprises a biological matrix. In some aspects, the biological sample comprises a cell lysate. In some aspects, the biological sample comprises a biofluid sample. In some aspects, the biofluid comprises blood, plasma, serum, urine, cerebrospinal fluid, or saliva. In some aspects, the biological sample is from a subject. In some aspects, the subject is a mammal. In some aspects, the subject is a human. In some aspects, the delivery moiety comprises an antibody. In some aspects, the first arm or the second arm is connected to the central moiety at least in part by a cleavable moiety. In some aspects, the third arm is connected to the central moiety at least in part by a cleavable moiety.
  • the cleavable moiety is photocleavable or enzymatically cleavable.
  • the cleavable moiety comprises an o-nitrobenzyloxy group, o-nitrobenzylamino group, o- nitrobenzyl group, o-nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, or pivaloyl group.
  • the cleavable moiety comprises an o-nitrobenzyl group substituted with a methoxy group or an ethoxy group.
  • the cleavable moiety is represented by the formula:
  • FIG. 1 illustrates an example of a multifunctional molecule.
  • FIG. 2 illustrates a specific example of a composition comprising a multifunctional molecule.
  • FIG. 3 illustrates an example of an enrichment method using a multifunctional molecule.
  • FIG. 4 illustrates a non-limiting example of a sketch of a multifunctional molecule described herein, where the multifunctional molecule comprises at least one delivery moiety, at least one react group, a central moiety, and at least one enrichment handle.
  • the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond.
  • the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one uncleavable bond.
  • the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond or at least one uncleavable bond. In some aspects, the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond and at least one uncleavable bond.
  • FIG. 5A-5F illustrate non-limiting examples of the multifunctional molecule described herein showing different arrangements of the cleavable bond.
  • FIG. 6 illustrates a non-limiting example of utilizing a multifunctional molecule described herein for an application of analyzing a targeted analyte.
  • FIG. 7 illustrates a non-limiting example of a multifunctional molecule described herein, where the reactive group, cleavable bond moiety, enrichment moiety, and the delivery moiety are highlighted.
  • FIG. 8 illustrates another non-limiting example of a multifunctional molecule described herein, where the reactive group, cleavable bond moiety, enrichment moiety, and the delivery moiety are highlighted.
  • FIG. 9 illustrates a flowchart showing how to utilize a multifunctional molecule described herein for enriching at least one macromolecule from a biological matrix obtained from a subject.
  • FIG. 10 illustrates a specific example of a composition comprising a multifunctional molecule and a protecting group on the multifunctional molecule.
  • FIG. 11 illustrates SEQ ID NO. 1 with the underlined amino acids designated as peptides found via LC-MS/MS analysis of glycosylated proteins bound to trifunctional molecules.
  • FIG. 12 illustrates a mass spectrometry of a protein.
  • FIG. 13 illustrates a mass spectrometry of a protein.
  • Enrichment of macromolecules may be useful for measuring amounts of the macromolecules.
  • proteins with a particular post translational modification (PTM) may be enriched prior to measurement of the proteins.
  • Protein glycosylation is an example of a protein PTM that plays a role in numerous biological and disease pathways. Gaining a deeper understanding of global protein glycosylation is useful understanding disease presence and/or progression. A challenge is that protein glycosylation is often difficult to characterize and quantify in many experimental workflows due to their complexity and abundance with respect to other biological compounds. A solution to overcome this difficulty is to enrich glycoproteins or glycopeptides prior to analysis.
  • the methods and compositions such as a multifunctional molecule described herein may be useful for specifically enriching and studying macromolecules such as glycoproteins and glycopeptides.
  • Disclosed herein are multifunctional molecules.
  • the multifunctional molecule may include a central moiety.
  • the multifunctional molecule may include a first arm comprising a delivery moiety connected to the central moiety, a second arm comprising a reactive group connected to the central moiety, or a third arm comprising an enrichment handle connected to the central moiety, or a combination thereof.
  • the first arm, the second arm, or the third arm may comprise a cleavable moiety, such as a photocleavable moiety.
  • the multifunctional molecule may be useful for a method described herein, such as for specifically enriching glycoproteins or other macromolecules from complex biological matrices. Some such molecules may be used to globally profile any or all glycoproteins from a complex biological matrix.
  • the method may include providing a multifunctional molecule.
  • the method may include contacting a sample comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule.
  • the method may be used to enrich the macromolecules in a biological matrix or fluid.
  • a surprising aspect of some embodiments is flexibility of the molecule to attach a delivery agent with ease via a click reaction to the delivery moiety.
  • the click reaction may be any copper-free click reactions between an azide and DBCO.
  • the delivery agent is azide-modified and the delivery moiety comprises DBCO.
  • the delivery agent comprises DBCO and the delivery moiety is azide- modified. Any copper-free click reactions between an azide and DBCO, for example, may be performed at similar reaction conditions, so linkage of any azide modified or DBCO modified delivery agent to a multifunctional molecule may be performed.
  • the linker comprises an alkylene.
  • the alkylene is a C1-C20 alkylene or a derivative thereof.
  • the C1-C20 alkylene may optionally be substituted variants thereof.
  • the alkylene is a C1-C10 alkylene or a derivative thereof.
  • the linker comprises an heteroalkylene.
  • the heteroalklyene comprises a PEGi- n , wherein n is any suitable integer.
  • n is an integer from 2-100. In some instances, n is an integer from 2-50. In some instances, n is an integer from 2-25. In some instances, n is an integer form 2-20.
  • the heteroalkylene comprises a PEGi-20 (e.g. 1 to 2+0.8+0.320 units of polyethene glycol) or a derivative thereof. In some instances, the PEG1-20 may optionally be substituted variants thereof. In some examples, the heteroalkylene comprises a PEG4 or a derivative thereof.
  • the linker may be modified, for example, with a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, or a carbonate.
  • the number of PEG units in a PEG linker or carbon atoms in an alkylene linker can be decreased or increased as needed. Varying the number of PEGs or carbon atoms in the linker may have varying effects chemical reactive arm reach. For example, longer PEG arms may be useful for allowing greater promiscuity, while and shorter PEG arms may provide more specificity.
  • a benefit of the compositions and methods herein may be that a macromolecule (e.g. protein or a glycoprotein) may be kept in tact during the enrichment process.
  • a glycosylation site may be identified on the protein or proteins.
  • Some aspects relate to a method of assaying or enriching glycoproteins while keeping the proteins in tact. Some aspects include identifying a site of glycosylation of any of the glycoproteins.
  • a multifunctional molecule is a trifunctional molecule.
  • a multifunctional molecule may include several features.
  • the multifunctional molecule may include multiple features useful for global profiling of glycosylated proteins, which may be used simultaneously in a single molecule for the specific enrichment of glycosylated proteins/peptides.
  • a multifunctional molecule or a trifunctional molecule may include all 3 of the aforementioned features, or a multifunctional molecule may include 1 or 2 of the following features.
  • a first feature may include a delivery moiety which can facilitate specific linkage of a delivery agent to the multifunctional molecule using click chemistry.
  • the delivery moiety may comprise a dibenzocyclooctyne (DBCO) moiety and the delivery agent may be azide-modified, which may be linked through copper-free click chemistry.
  • DBCO dibenzocyclooctyne
  • a second feature may include a reactive group such as a hydrazide reactive group.
  • the active molecule may be brought into close proximity to a glycosylated protein or peptide by the delivery agent.
  • the glycosylated protein or peptide may, for example, be brought into close proximity with the delivery agent via electrostatic interactions.
  • the interaction may comprise van der Waals forces.
  • the interaction may comprise dipole-dipole interactions.
  • a third feature of a multifunctional molecule may include an enrichment handle such as a biotin or desthiobiotin enrichment handle.
  • an enrichment handle such as a biotin or desthiobiotin enrichment handle.
  • the entire multifunctional molecule-gly coprotein or glycopeptide complex can be captured by affinity enrichment with an avidin or streptavidin solid support. Proteins or peptides that do not have glycosylation sites, may then be washed away after not being bound on the avidin or streptavidin support. The glycosylated proteins or peptides retained on the support after washing may thereby be significantly and specifically enriched, and can then be prepared for analysis assay procedures and.
  • the multifunctional molecule may be represented by the formula:
  • the central moiety may be represented by A.
  • the central moiety may include a heterocycle, a carbocycle, or a trivalent nitrogen.
  • the trivalent nitrogen may include an amine.
  • the amine may include a tertiary amine.
  • Any of the aforementioned features may be connected to the central moiety via a linker (L 1 , L 2 , and L 3 ).
  • R 1 may comprise the delivery moiety
  • R 2 may comprise the reactive group
  • R 3 may comprise the enrichment handle.
  • the linker may optionally include a cleavable moiety, such as those described herein.
  • the linker may optionally include a connecting group, such as those described herein.
  • the delivery moiety may include an alkyne.
  • the alkyne is a terminal alkyne.
  • the alkyne is a cyclic alkyne.
  • the delivery moiety may be ring strained, for example by including a triple bond which is stable when linear.
  • the ring strained delivery moiety may be bent in a non-linear form.
  • the ring strained delivery moiety may be reactive.
  • the delivery moiety is a cyclic alkyne. Cyclic alkyne groups may be useful for the present invention because it allows for azide-modified agents to be attached to the multifunctional group through click chemistry.
  • the delivery moiety is a DBCO moiety.
  • a delivery moiety excludes an amine reactive crosslinker.
  • the delivery moiety may be activated with a delivery agent that binds the macromolecule.
  • the delivery agent may be azide-modified.
  • the delivery moiety and the delivery agent may react via copper-free click chemistry (e.g., when the delivery moiety comprises DBCO).
  • the delivery moiety and the delivery agent may react via copper-catalyzed click chemistry (e.g., when the delivery moiety comprises a terminal alkyne).
  • a delivery agent may be useful for bringing a macromolecule into proximity with the multifunctional molecule.
  • the delivery agent covalently bound to the multifunctional molecule may bind non-covalently with the macromolecule.
  • Non-covalent binding of the macromolecule with the activated delivery moiety may bring the macromolecule into proximity with a reactive group of the multifunctional molecule, so that the reactive group may bind covalently to the macromolecule.
  • a multifunctional molecule that includes a delivery moiety comprising DBCO reacted with a delivery agent such as azide-modified affinity reagent (such as a lectin) may be able to bind macromolecules that include sugars.
  • the functional groups involved in the copper-free click reaction may be interchanged.
  • the delivery moiety may be azide-modified and the delivery agent may comprise a DBCO moiety.
  • the delivery agent may comprise an amphiphilic polymer.
  • the delivery agent may comprise a lecithin.
  • the lecithin may comprise one or more glycerophospholipids.
  • the one or more glycerophospholipids may comprise phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or phosphatidic acid.
  • the first arm may be a linker L 1 having the formula: -X ⁇ L ⁇ -CM ⁇ L ⁇ -Y 1 -*, wherein * represents the connection to the delivery moiety.
  • CM 1 may be a cleavable moiety, such as those described herein.
  • CM 1 may be absent.
  • L la and L lb may each independently be an alkylene, a heteroalkylene, or an optionally substituted variant thereof, such as those described herein.
  • L la or L lb may each independently is a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety or the cleavable moiety.
  • PEG linker of the first arm may include PEG2-8.
  • the PEG linker of the first arm may include PEG4.
  • L la and L lb may each independently be absent.
  • X 1 and Y 1 may each independently be a connecting group.
  • X 1 and Y 1 may each independently be absent.
  • the connecting group of X 1 and Y 1 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO 2 -, - C(O)Ci-C 10 alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-, -C1-C10 alkyl-CO 2 -, -C1-C10 alkyl-NH-, -C1-C10 alkyl-O-
  • the connecting group of X 1 and Y 1 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
  • the reactive group may include a hydrazide.
  • the hydrazide may react with an oxidized sugar (e.g. aldehyde or ketone) on a glycosylated protein or a glycosylated peptide to form a hydrazone bond between the reactive group and the glycosylated protein or the glycosylated peptide.
  • This may also be lipids or metabolites or other small molecules with an aldehyde functional group.
  • the second arm may be a linker L 2 having the formula: -X 2 -L 2a -CM 2 -L 2b -Y 2 -*, wherein * represents the connection to the reactive group.
  • CM 2 may be a cleavable moiety, such as those described herein.
  • CM 2 may be absent.
  • L 2a and L 2b may each independently be an alkylene, a heteroalkylene, or a optionally substituted variant thereof, such as those described herein.
  • L 2a or L 2b may each independently is a polyethylene glycol (PEG) linker connecting the reactive group to the central moiety or the cleavable moiety.
  • PEG polyethylene glycol
  • the PEG linker of the first arm may include PEG2-8.
  • the PEG linker of the first arm may include PEG4.
  • L 2a and L 2b may each independently be absent.
  • X 2 and Y 2 may each independently be a connecting group.
  • X 2 and Y 2 may each independently be absent.
  • the connecting group of X 2 and Y 2 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO 2 -, - C(O)Ci-C 10 alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-, -C1-C10 alkyl-CO 2 -, -C1-C10 alkyl-NH-, -C1-C10 alkyl-O-
  • the connecting group of X 2 and Y 2 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
  • the enrichment handle may include biotin, desthiobiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof.
  • the enrichment handle may include biotin.
  • the enrichment handle may include desthiobiotin.
  • the enrichment handle may bind to an affinity reagent.
  • the affinity reagent may be connected to a solid support.
  • the affinity reagent may include avidin.
  • the affinity reagent may include streptavidin.
  • the solid support may include a bead, such as a bead in solution.
  • the macromolecule when bound with the multifunctional molecule may be enriched by binding the enrichment handle with any type of solid support, for example, streptavidin coated glass plates to streptavidin coated beads.
  • the solid support may be suspended in the sample and then spun down into a pellet bringing the bound macromolecule, while non-enriched macromolecules not bound to multifunctional molecules remain in solution in the sample.
  • the enrichment handle is directly connected to a solid support.
  • the multifunctional molecule may include a third arm comprising an enrichment handle connected to the central moiety.
  • the second arm may be a linker L 3 having the formula: -X 3 -L 3a -CM 3 -L 3b -Y 3 -*, wherein * represents the connection to the enrichment handle.
  • CM 3 may be a cleavable moiety, such as those described herein.
  • CM 3 may be absent.
  • L 3a and L 3b may each independently be an alkylene, a heteroalkylene, or an optionally substituted variant thereof, such as those described herein.
  • L 3a or L 3b may be a C1-C10 alkylene or a derivative thereof.
  • L 3a or L 3b may be absent.
  • X 3 and Y 3 may each independently be a connecting group. In some instances, X 3 and Y 3 , may each independently be absent. In some cases, the connecting group of X 3 and Y 3 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO 2 -, - C(0)Ci-Cio alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-,
  • the connecting group of X 3 and Y 3 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
  • the linker of any of the arms may include an alkylene.
  • the alkylene may include a Ci-C 2 o alkylene or a derivative thereof.
  • the alkylene may include a Ci alkylene, a C 2 alkylene, a C3 alkylene, a C4 alkylene, a C5 alkylene, a Ce alkylene, a C7 alkylene, a Cx alkylene, a C9 alkylene, a C10 alkylene, a Cn alkylene, a Ci 2 alkylene, a C13 alkylene, a C14 alkylene, a C15 alkylene, a Ci6 alkylene, a C17 alkylene, a Cis alkylene, a C19 alkylene, a C 2 o alkylene, or any range thereof.
  • Peptide or peptide-like backbones may be used in a linker or connecting arm.
  • the linker may include an amine - alkyl (e.g. side chain attachment) - carbonyl repeat, or a component thereof.
  • the linker may include an amine.
  • the linker may include an alkyl.
  • the linker may include a carbonyl.
  • a biotin may be linked to a central amine directly through a reaction between the amine and biotin.
  • some aspects do not include a PEG linker in this part of the multifunctional molecule.
  • a central amine may be reacted with a carboxylic acid of the biotin.
  • the third arm may include a PEG linker connecting the enrichment handle to the central moiety.
  • there may be an alcohol group that forms a bond with biotin.
  • a trifunctional molecule may include a central tertiary amine that has three linker arms, each housing a different chemical functionality.
  • n may be 2-8.
  • n may be 4.
  • m may be 2-8.
  • m may be 4.
  • a trifunctional molecule may include a central tertiary amine that has three linker arms, each housing a different chemical functionality, wherein at least one of the three linkers may comprise a cleavable moiety. In some aspects, at least two of the three linkers may comprise a cleavable moiety. In some aspects, all three of the linkers may comprise a cleavable moiety. In some instances, the cleavable moiety may be between the central moiety and a reactive moiety (e.g., hydrazide), such as in the exemplary formula represented by the structure:
  • a reactive moiety e.g., hydrazide
  • the cleavable moiety may between the central moiety and the delivery moiety (e.g., DBCO), such as in the exemplary formula represented by the structure:
  • a cleavable moiety may be cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof.
  • the light may comprise UV light, visible light, IR light, laser, or a combination thereof.
  • the cleavable moiety may be a photocleavable moiety.
  • the photocleaveable moiety may comprise an electron withdrawing group, such as, but not limited to a nitro group or halide group.
  • the cleavable moiety may be an enzymatically cleavable moiety.
  • the cleavable moiety may include a pH sensitive cleavable bond which can be cleaved under acidic or basic conditions.
  • the cleavable moiety may include a pH sensitive cleavable bond which is cleaved by acidifying the solution.
  • the cleavable moiety may include a pH sensitive cleavable bond which is cleaved by making the solution more basic. The pH sensitive cleavable bond is advantageous because the molecule can be delivered, but would not react until it was under a slightly acidified environment which can be beneficial for drug delivery.
  • the cleavable moiety may include a disulfide bond.
  • the disulfide bond may be chemically or enzymatically formed.
  • the disulfide bond may be cleaved by a reducing agent.
  • the disulfide bond may be enzymatically cleavable.
  • the cleavable moiety may include a protein or peptide sequence that is recognized and cleaved by the enzyme.
  • the cleavable moiety may include the peptide sequence ENLYFQ*S (where * denotes a cleavage site).
  • the disulfide bond may be included as part of a peptide.
  • An enzyme that cleaves a cleavable moiety may include an enzyme that cleaves a disulfide bond. Some examples of enzymes that may cleave disulfide bonds include thioredoxin or glutaredoxin. The enzyme may include trypsin. The enzyme may include a virus that cleaves a specific peptide sequence. For example, a tobacco etch virus (TEV) protein that specially cleaves the peptide sequence ENLYFQ*S (where * denotes a cleavage site) may be used. This or another peptide sequence may be present in between the central moiety and one (or any) of the arms. After linkage and enrichment, may bond could be cleaved, thereby releasing the molecule of interest.
  • TSV tobacco etch virus
  • the photocleavable moiety in the trifunctional molecule may be cleaved by UV light.
  • the UV light may have a wavelength in the range of about 100 nm to about 400 nm, about 200 nm to about 400 nm, about 250 nm to about 400 nm, about 280 nm to about 400 nm, about 100 nm to about 370 nm, about 200 nm to about 370 nm, about 250 nm to about 370 nm, or about 280 nm to about 370 nm.
  • the photocleavable moiety comprises a nitrobenzyl oxy group, nitrobenzylamino group, nitrobenzyl group, nitroveratryl group, phenacyl group, alkoxyphenacyl group, benzoin group, or a pivaloyl group.
  • the nitro group may be in the ortho position of the benzyl, veratryl, phenacyl, benzoin, or pivaloyl group relative to site of cleavage (e.g., o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o-nitroveratryl group).
  • the alkoxy group may be in the para position of the benzyl, veratryl, phenacyl, benzoin, or pivaloyl group relative to the site of cleavage (e.g., p-alkoxyphenacyl group).
  • the photocleavable moiety comprises a nitrobenzyl group.
  • the nitro group may be ortho to the benzyl group relative to the site of cleavage (o-nitrobenzyl group).
  • the o-nitrobenzyl group may be substituted with a methoxy or an ethoxy. In some cases, the methoxy or ethoxy may be substituted in the para position relative to the nitro of the o-nitrobenzyl group.
  • the o-nitrobenzyl group may comprise a linkage connecting to a linker, such as those described herein, that further connects to the central moiety.
  • the linkage may be in the meta position relative to the nitro group.
  • the linkage may comprise an ester, an ether, an amine, an amide, a carbamate, -O- Ci-Cio alkyl-, or any other linkage described herein.
  • the photocleavable moiety may comprise the structure represented by the formula: such examples, n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, n may be 3.
  • the photocleavable moiety in the trifunctional molecule may be cleaved by a laser.
  • the laser is may be in the UV ranges.
  • the UV range may be those described herein.
  • the photocleavable moiety comprises a benzyl halide.
  • the halide group may comprise a fluoride, chloride, iodide, bromide, or a combination thereof.
  • the photocleavable moiety comprises at least one, at least two, at least three, or at least four halide groups.
  • the photocleavable moiety comprises no more than one, no more than two, no more than three, or no more than four halide groups.
  • the halide group is a iodide.
  • the iodide may be in the ortho position of the benzyl group relative to site of cleavage.
  • the benzyl halide may comprise a linkage connected to a linker, such as those described herein, that further connects to the central moiety.
  • the linkage may be in the meta position relative to the halide.
  • the linkage may comprise any linkage structure or connecting structure described herein.
  • the multifunctional molecule may include a protecting group.
  • the multifunctional molecule may be deprotected.
  • some aspects include providing a multifunctional molecule including a protecting group, and deprotecting the multifunctional molecule.
  • a reactive moiety of the multifunctional molecule may include the protecting group.
  • An example may include a boc-hydrazide.
  • the multifunctional molecule may be included in a composition.
  • the composition may include a solution.
  • the multifunctional molecule may be included in the solution as a salt.
  • the solution may include a counterion such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the counter ion may be acetic acid.
  • the counter ion may be triethyl ammonium.
  • methods that include enrichment of a macromolecule.
  • the enrichment may be performed using a multifunctional molecule.
  • Some aspects include providing a multifunctional molecule.
  • the multifunctional molecule may be provided in solution.
  • An enrichment method may include solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule.
  • the multifunctional molecule may be provided as a salt.
  • the multifunctional molecule may be provided with a counterion, such as those described herein.
  • the counterion may comprise trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the counterion does not include hydrochloric acid (HCL).
  • HCL may, in some cases, degrade the multifunctional molecule.
  • An enrichment method may include activating click chemistry, such as copper-free or copper-mediated (e.g., Cu(I)) click chemistry.
  • the click chemistry may be between the delivery moiety and a delivery agent. The activation may be before contacting the sample with the multifunctional molecule.
  • the method may include activating copper-free click chemistry between the delivery moiety and the delivery agent prior to contacting the sample with the multifunctional molecule.
  • the delivery moiety may comprise DBCO and the delivery agen may comprise an azide. Alternatively, the delivery moiety may comprise an azide and the delivery agent may comprise a DBCO. Copper free click chemistry between DBCO and an azide group may take place at room temperature with gentle mixing resulting in an “active” molecule.
  • the “active” version of the trifunctional molecule may be mixed at specific ratios with a biological matrix such as plasma, serum, or a cell lysate.
  • An azide modified delivery agent and a delivery moiety such as DBCO may be reacted to produce an activated delivery moiety (comprising the delivery agent).
  • a reaction may take place by including a mixture of an azide modified delivery agent and DBCO in ethanol and water. This reaction may reach completion after approximately an hour at room temperature with gentle mixing. Mixing may include agitation. Reaction completion can be modified by monitoring absorbance values at 290 nm. This wavelength may be characteristic of DCBO and may disappear or be reduced once the reaction is completed.
  • the active trifunctional molecule may they be mixed with a sample.
  • An incubation time for contacting the biological matrix and trifunctional molecule may be a few hours, and the incubation may be performed at either cold (4 degrees Celsius) or body temperature (37 degrees Celsius). Washing conditions that may be used may be quite stringent since some glycosylated macromolecules may be covalently linked to the trifunctional molecule, and such washing conditions may be useful for specifically enriching glycosylated macromolecules.
  • the enriched macromolecules may then undergo sample preparation steps for assaying the enriched macromolecules.
  • a delivery agent may be selected based upon the macromolecule to be enriched.
  • the selected delivery agent may be azide-modified before or as part of a method described herein.
  • the azide modification may enable reaction and binding of the delivery agent to the delivery moiety (for example, to a delivery moiety comprising DBCO).
  • a protein can be used as the delivery reagent.
  • the delivery agent may include an antibody.
  • the delivery agent may include any protein or antibody that binds to the macromolecule to be enriched, or a binding fragment thereof.
  • An example of a protein delivery agent includes a lectin protein.
  • the lectin protein may bind glycans. The binding of the lectin protein to a glycan may be specific.
  • the delivery agent may include a commercially available antibody or protein.
  • the antibody may be customized (e.g. non-commercial).
  • Another example of delivery a agent may include an aptamer.
  • the aptamer may be azide modified.
  • the aptamer may be specific to a macromolecule (e.g. protein or type of protein such as glycoprotein).
  • the aptamer may include DNA.
  • the aptamer may include RNA.
  • the aptamer may be modified or include nucleotide modifications.
  • Another example of a delivery agent may include a nanoparticle.
  • the nanoparticle may bind to a macromolecule of interest.
  • the delivery agent may include a protein, antibody, aptamer, or nanoparticle.
  • the delivery agent may undergo azide introduction to modify the delivery agent (e.g. an antibody or lectin).
  • the azide-modified agent may then be reacted with an alkyne (e.g. terminal or DBCO).
  • the delivery agent may be modified with an azide though routine synthesis, and then attached to the trifunctional molecule and be ready for macromolecule enrichment.
  • the antibody may undergo azide introduction to modify the antibody.
  • a lectin may be modified with an azide though routine synthesis, then can be attached to the trifunctional molecule and ready for glycan enrichment.
  • Use of an antibody would follow a very similar process in that it would be modified with an azide transfer then would be reacted with the alkyne in a separate second click step.
  • the enrichment method may include reacting a delivery agent to the DBCO.
  • the delivery agent may include any type of molecule that brings the multifunctional molecule to a target macromolecule.
  • the delivery agent may include an azide-modified lectin.
  • a lectin may be useful for binding sugars or glycoproteins.
  • the delivery agent may click onto the multifunctional molecule.
  • the multifunctional molecule activated with the delivery agent may be mixed with a sample such as a biological matrix. In the case of the lectin, the lectin may bring the multifunctional molecule to a glycoprotein, where a reactive group (e.g. a hydrazide) of the multifunctional molecule links to the target macromolecule covalently.
  • a reactive group e.g. a hydrazide
  • the glycosylated protein or peptide may, for example, be brought into close proximity with the delivery agent via electrostatic interactions, such as van der Waals forces or dipole-dipole interactions.
  • the delivery agent comprises an amphiphilic polymer.
  • the multifunctional molecule linked to the target macromolecule may be enriched, and have non-linked macromolecules washed away.
  • the delivery agent may include an alkyne.
  • the alkyne may be a terminal alkyne.
  • Some aspects include activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule.
  • Activating the click chemistry may include gentle mixing.
  • Biological samples may be treated with under mild or moderate oxidation conditions, for example, to oxidize either terminal or both terminal and internal sugars. This may result in aldehydes that will react with the hydrazide moiety on our molecule.
  • the method may include oxidizing the sample, thereby oxidizing the macromolecule, prior to contacting the sample with the multifunctional molecule.
  • the method may include oxidizing internal or external sugars of the macromolecule. Oxidizing the macromolecule may generates an aldehyde of the macromolecule. Oxidizing the macromolecule may generates an aldehyde of the internal or external sugars of the glycosylated macromolecule.
  • oxidizers of a sugar may include, but are not limited to sodium periodate (NalOs), MnO, C2CI2O2, KC Ch, K2CrO4, dess-martin periodinane (CBHBIOS), or perruthenate (e.g., N(C3H?)4RuO4).
  • the aldehyde may react with the reactive group.
  • the oxidizer may comprise sodium peroxidate.
  • Terminal glycans can be oxidized under mild concentration of NalCh. Additional, both terminal and internal sugar groups can be oxidized with a higher concentration of NalCh. Examples of mild concentration of NalCh includes, but is not limited to about 1.0 mM of NalCh, 1.5 mM of NalCh, 2.0 mM of NalCE, and 2.5 mM of NalCh. Examples of higher concentration of NalCh includes but is not limited to about 10 mM of NalCh, about 15 mM of NalCh, and about 20 mM of NalCh.
  • the method may include binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule.
  • the affinity reagent may be connected to a solid support.
  • the affinity reagent may include avidin.
  • the affinity reagent may include streptavidin.
  • the method may include further comprising concentrating the captured multifunctional molecule bound to the macromolecule.
  • the method may include precipitating, centrifuging, eluting, or a combination thereof.
  • the macromolecule may then be released and assayed. In some cases, only part of the macromolecule is released or assayed.
  • the method may include releasing at least part of the macromolecule from the multifunctional molecule.
  • the macromolecule may be processed while captured on a solid support such as a bead, and just part of the macromolecule may be released and assayed in some cases.
  • the method may include assaying the macromolecule.
  • the assaying may include performing mass spectrometry (MS), chromatography, liquid chromatography (LC), high- performance liquid chromatography, solid-phase chromatography, a lateral flow assay, an immunoassay, an enzyme-linked immunosorbent assay, a western blot, a dot blot, or immunostaining, or a combination thereof.
  • the assaying may include performing MS.
  • MS may be used to assay proteins or other such as metabolites or lipids.
  • the assaying may include LC-MS.
  • Assaying may include use of a protein microarray.
  • assaying may include performing nucleic acid sequencing, microarray analysis, hybridization, polymerase chain reaction (PCR), or electrophoresis, or a combination thereof. Assaying may include use of a microarray. Assaying may include use of a nucleic acid microarray. [0070] The assaying may be used to generate assay results.
  • the assay results may include macromolecule measurements.
  • the macromolecule measurements may include protein measurements such as glycoprotein measurements, or a measurement of a protein with a post- translational modification.
  • Some aspects include generating the macromolecule measurements. Some aspects include receiving the macromolecule measurements. For example, macromolecule measurements may be received after any of the methods described herein have been performed. [0072] The macromolecule measurements may be useful for identifying a biological state.
  • the method may include identifying the macromolecule in the sample as indicative of a biological state.
  • the biological state may include a disease state.
  • the biological state may include a healthy state.
  • the method may include identifying the subject as having the biological state.
  • a healthcare professional receives the macromolecule measurements, or a report based on the macromolecule measurements, and diagnosis the subject as having a disease state or identifies the subject as having a healthy biological state.
  • Some aspects include providing a treatment to the subject based on the disease state. For example, a treatment may include administration of a pharmaceutical composition.
  • the method may be used to generate results for multiple macromolecules.
  • the measurements may include measurements of many proteins (e.g. glycoproteins), which may be used in identifying the subject has having the biological state, or in identifying the sample as indicative of the biological state.
  • the measurements may include an omic type of measurement.
  • mass spectrometry may be used to identify all proteins (or other macromolecules) in a sample after enrichment of the sample for the macromolecules.
  • all proteins that have a certain post translational modification such as glycosylation are enriched and measured together using a mass spectrometry or other technique. Then, the measurements may be used as indicated.
  • modified or engineered macromolecule such as an engineered protein.
  • the modified or engineered macromolecule may be coupled to a compound described herein.
  • the coupling may include a bond.
  • the bond may be covalent.
  • the macromolecule may include a protein, carbohydrate, lipid, metabolite or nucleic acid.
  • the macromolecule may include a protein.
  • the macromolecule may include a carbohydrate.
  • the macromolecule may include a lipid.
  • the macromolecule may include a metabolite.
  • the macromolecule may include a nucleic acid.
  • the macromolecule may include a protein with a post-translational modification.
  • the post-translational modification may include glycosylation.
  • the protein may include a glycoprotein.
  • the protein may include a proteoglycan.
  • the protein may include a glycosylated protein.
  • the sample may include a biological sample.
  • the biological sample may include a biological matrix.
  • the biological sample may include a cell lysate.
  • the biological sample or biological matrix may include a biofluid sample.
  • the sample may include a biofluid.
  • the biofluid may include blood, plasma, serum, urine, cerebrospinal fluid, or saliva.
  • the biofluid sample may include a blood sample.
  • the biofluid sample may include a plasma sample.
  • the biofluid sample may include a serum sample.
  • the biofluid sample may include a urine sample.
  • the biofluid sample may include a cerebrospinal fluid sample.
  • the biofluid sample may include a saliva sample.
  • biological samples include, but are not limited to, plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, or cell culture samples.
  • the biofluid is plasma or serum.
  • the biofluid is cerebrospinal fluid.
  • the subject may be a vertebrate.
  • the subject may be a mammal.
  • the mammal may include a rat, mouse, gerbil, guinea pig, or hamster.
  • the mammal may include a fox, bear, dog, monkey, cow, pig, or sheep.
  • the subject may be a primate.
  • the primate may include an ape or monkey.
  • the primate may include a chimpanzee, a lemur, a bonobo, an orangutan, or a baboon.
  • the subject may be a human.
  • the subject may be an adult (e.g. at least 18-years-old).
  • the subject may be male.
  • the subject may be female.
  • the subject may have a disease state.
  • the subject may have a disease or disorder, a comorbidity of a disease or disorder, or may be healthy.
  • a method consistent with the present disclosure may comprise collecting (e.g., isolating, enriching, or purifying) a species from biological sample.
  • the species may be a biomolecule (e.g., a protein), a biomacromolecular structure (e.g., a peptide aggregate or a ribosome), a cell, or tissue.
  • the species may be selectively collected from the biological sample.
  • a method may comprise isolating cancer cells from tissue (e.g., as a tissue biopsy) or from a biofluid (e.g., as a liquid biopsy) such as whole blood, plasma, or a buffy coat.
  • the method may include a sample without cancer cells.
  • the species may be treated prior to analysis. For example, a protein may be reduced and degraded, a nucleic acid may be separated from histones, or a cell may be lysed.
  • the biological samples may be obtained or derived from a human subject.
  • the biological samples may be stored in a variety of storage conditions before processing, such as different temperatures (e.g., at room temperature, under refrigeration or freezer conditions, at 25°C, at 4°C, at -18°C, -20°C, or at -80°C) or different suspensions (e.g., EDTA collection tubes).
  • the methods described herein may be used to evaluate a disease state or a biological state.
  • the methods described herein may be used to predict or identify a biological state.
  • the methods described herein may be used to predict or identify a disease state, or a lack thereof.
  • a treatment may be provided to individuals having a disease state.
  • a disease state may include a disease or disorder such as cancer. Examples of cancer include lung cancer, colon cancer, pancreatic cancer, liver cancer, ovarian cancer, breast cancer, prostate cancer, melanoma, bladder cancer, lymphoma, leukemia, renal cancer, or uterine cancer. In some aspects, the cancer is breast cancer.
  • a disease may include a disorder.
  • a disease state may include having a comorbidity related to a disease or disorder.
  • a reference to whether a subject has a disease state or not may include the subject being healthy.
  • a healthy state may exclude a disease state.
  • a healthy state may exclude having cancer.
  • a disease state may exclude being healthy.
  • the methods may be useful for cancer diagnosis.
  • the methods may be useful for cancer screening.
  • the method may be useful for cancer treatment.
  • the classifier is generated using proteomic data obtained by contacting training samples with particles such that the particles adsorb proteins in the training samples, and assaying the proteins adsorbed to the particles. Some aspects include obtaining of receiving the biofluid sample of the subject.
  • the cancer may be at an early stage or a late stage.
  • An example of an early stage of cancer may include stage I.
  • An early stage may include stage I or II.
  • An early stage may include stage I, II, or III.
  • An example of late stage cancer may include stage 4.
  • the enrichment of macromolecules with the multifunctional molecules described herein may be used to data determination and analysis.
  • the data described herein may include protein data or proteomic data.
  • Proteomic data may involve data about proteins, peptides, or proteoforms. This data may include just peptides or proteins, or a combination of both.
  • An example of a peptide is an amino acid chain.
  • An example of a protein is a peptide or a combination of peptides.
  • a protein may include one, two or more peptides bound together.
  • a protein may be a secreted protein.
  • Proteomic data may include data about various proteoforms.
  • proteoforms can include different forms of a protein produced from a genome with any variety of sequence variations, splice isoforms, or post-translational modifications.
  • the proteomic data may be generated using an unbiased, non-targeted approach, or may include a specific set of proteins. Aspects described in relation to proteomic data may be relevant to protein data, or vice versa.
  • Proteomic data may include information on the presence, absence, or amount of various proteins and peptides.
  • proteomic data may include amounts of proteins.
  • a protein amount may be indicated as a concentration or quantity of proteins, for example a concentration of a protein in a biofluid.
  • a protein amount may be relative to another protein or to another biomolecule.
  • Proteomic data may include information on the presence of proteins or peptides.
  • Proteomic data may include information on the absence of proteins or peptides.
  • Proteomic data may be distinguished by subtype, where each subtype includes a different type of protein, peptide, or proteoform.
  • proteomic data generally includes data on a number of proteins or peptides.
  • proteomic data may include information on the presence, absence, or amount of 1000 or more proteins or peptides.
  • proteomic data may include information on the presence, absence, or amount of 5000, 10,000, 20,000, or more peptides, proteins, or proteoforms.
  • Proteomic data may even include up to about 1 million proteoforms.
  • Proteomic data may include a range of proteins, peptides, or proteoforms defined by any of the aforementioned numbers of proteins, peptides, or proteoforms.
  • the data comprises measurements of over 10 peptides or protein groups, over 15 peptides or protein groups, over 20 peptides or protein groups, over 25 peptides or protein groups, over 30 peptides or protein groups, over 35 peptides or protein groups, over 40 peptides or protein groups, over 45 peptides or protein groups, over 50 peptides or protein groups, over 75 peptides or protein groups, over 100 peptides or protein groups, over 250 peptides or protein groups, over 500 peptides or protein groups, over 1,000 peptides or protein groups, over 2,500 peptides or protein groups, over 5,000 peptides or protein groups, over 10,000 peptides or protein groups, over 15,000 peptides or protein groups, or over 20,000 peptides or protein groups.
  • the data comprises measurements of at least about 10 peptides or protein groups, at least about 15 peptides or protein groups, at least about 20 peptides or protein groups, at least about 25 peptides or protein groups, at least about 30 peptides or protein groups, at least about 35 peptides or protein groups, at least about 40 peptides or protein groups, at least about 45 peptides or protein groups, at least about 50 peptides or protein groups, at least about 75 peptides or protein groups, at least about 100 peptides or protein groups, at least about 250 peptides or protein groups, at least about 500 peptides or protein groups, at least about 1,000 peptides or protein groups, at least about 2,500 peptides or protein groups, at least about 5,000 peptides or protein groups, at least about 10,000 peptides or protein groups, at least about 15,000 peptides or protein groups, or at least about 20,000 peptides or protein groups.
  • the protein data comprises measurements of no greater than 10 peptides or protein groups, no greater than 15 peptides or protein groups, no greater than 20 peptides or protein groups, no greater than 25 peptides or protein groups, no greater than 30 peptides or protein groups, no greater than 35 peptides or protein groups, no greater than 40 peptides or protein groups, no greater than 45 peptides or protein groups, no greater than 50 peptides or protein groups, no greater than 75 peptides or protein groups, no greater than 100 peptides or protein groups, no greater than 250 peptides or protein groups, no greater than 500 peptides or protein groups, no greater than 1,000 peptides or protein groups, no greater than 2,500 peptides or protein groups, no greater than 5,000 peptides or protein groups, no greater than 10,000 peptides or protein groups, no greater than 15,000 peptides or protein groups, or no greater than 20,000 peptides or protein groups.
  • the peptides or protein groups may comprise or consist
  • a protein may also include a post-translational modification (PTM).
  • PTM post-translational modification
  • An example of a PTM may include glycosylation. Proteins or peptides may include glycoproteins or glycopeptides. A protein may include a glycoprotein. A peptide may include a glycopeptide. An example of a PTM includes glycosylation. An example of a PTM may include phosphorylation. Proteins or peptides may include phosphoproteins or phosphopeptides. A protein may include a phosphoprotein. A peptide may include a phosphopeptide.
  • Proteomic data may be generated by any of a variety of methods. Generating proteomic data may include using a detection reagent that binds to a peptide or protein and yields a detectable signal. After use of a detection reagent that binds to a peptide or protein and yields a detectable signal, a readout may be obtained that is indicative of the presence, absence or amount of the protein or peptide. Generating proteomic data may include concentrating, filtering, or centrifuging a sample.
  • Proteomic data may be generated using mass spectrometry, chromatography, liquid chromatography, high-performance liquid chromatography, solid-phase chromatography, a lateral flow assay, an immunoassay, an enzyme-linked immunosorbent assay, a western blot, a dot blot, or immunostaining, or a combination thereof.
  • Some examples of methods for generating proteomic data include using mass spectrometry, a protein chip, or a reverse-phased protein microarray.
  • Proteomic data may also be generated using an immunoassay such as an enzyme-linked immunosorbent assay, western blot, dot blot, or immunohistochemistry assay. Generating proteomic data may involve use of an immunoassay panel.
  • One way of obtaining proteomic data includes use of mass spectrometry.
  • An example of a mass spectrometry method includes use of high resolution, two-dimensional electrophoresis to separate proteins from different samples in parallel, followed by selection or staining of differentially expressed proteins to be identified by mass spectrometry.
  • Another method uses stable isotope tags to differentially label proteins from two different complex mixtures. The proteins within a complex mixture may be labeled isotopically and then digested to yield labeled peptides. Then the labeled mixtures may be combined, and the peptides may be separated by multidimensional liquid chromatography and analyzed by tandem mass spectrometry.
  • a mass spectrometry method may include use of liquid chromatography-mass spectrometry (LC-MS), a technique that may combine physical separation capabilities of liquid chromatography (e.g., HPLC) with mass spectrometry.
  • LC-MS liquid chromatography-mass spectrometry
  • Proteins may be enriched prior to assaying or measuring them.
  • the enrichment may enrich one set of proteins and not another set, or may enrich a single protein and not another protein. Enrichment may be obtained through the use of an affinity reagent, for example by incubating the affinity reagent with a sample prior to measuring proteins in the sample.
  • the affinity reagent may include an antibody.
  • the affinity reagent may include a particle such as a nanoparticle. Proteins may be adsorbed to the affinity reagent, separated from the rest of the sample, and then assayed by using a proteomic assay described herein.
  • One aspect of generating proteomic data may include adsorbing proteins (or glycoproteins) to particles such as nanoparticles.
  • Samples may be contacted with particles, for example prior to generating data.
  • the data described herein may generated using particles.
  • a method may include contacting a sample with particles such that the particles adsorb biomolecules (e.g. macromolecules such as proteins).
  • the particles may attract different sets of biomolecules than would normally be measured accurately by performing an omics measurement directly on a sample.
  • a dominant biomolecule may make up a large percentage of certain type of biomolecules (e.g., proteins, transcripts, genetic material, or metabolites) in a sample.
  • one protein may make up a large portion of proteins in circulation that is collected by blood sampling.
  • biomolecules By adhering biomolecules to particles prior to analyzing the biomolecules, a subset of biomolecules may be obtained that does not include the dominant biomolecule. Removing dominant biomolecules in this way may increase the accuracy of biomolecule measurements and sensitivity of an analysis using those measurements.
  • Particles can be made from various materials. Such materials may include metals, magnetic particles, polymers, or lipids.
  • a particle may be made from a combination of materials.
  • a particle may comprise layers of different materials. The different materials may have different properties.
  • a particle may include a core comprising one material, and be coated with another material. The core and the coating may have different properties.
  • Generating proteomic data may include contacting a sample with particles such that the particles adsorb biomolecules comprising proteins.
  • the adsorbed proteins may be part of a biomolecule corona.
  • the adsorbed proteins may be measured or identified in generating the proteomic data.
  • Generating proteomic data may include the use of known amounts internal reference proteins.
  • the reference proteins may be labeled.
  • the label may include an isotopic label.
  • Generating proteomic data may include the use of known amounts of isotopically labeled, internal reference proteins (referred to as “PiQuant”).
  • the internal reference proteins may be spiked into a sample.
  • the internal reference proteins may be used to identify mass spectra of individual endogenous proteins.
  • the internal reference proteins may be used as standards for determining amounts of the individual endogenous proteins.
  • Proteomic measurements may be generated based on amounts of proteins added into a sample of the one or more biofluid samples.
  • Proteomic measurements may be generated based on amounts of labeled proteins added into a sample of the one or more biofluid samples.
  • the protein data or proteomic data may be obtained in conjunction with a method described herein such as an enrichment method.
  • protein data may be obtained after isolation of proteins comprising a particular subgroup such as a PTM using a method disclosed herein.
  • Protein data may be obtained using glycosylated proteins enriched using a method or composition disclosed herein.
  • proteomic data analysis may be carried out using a computer system.
  • a readout indicative of the presence, absence or amount of a biomolecule e.g., proteins such as glycoproteins
  • the computer system may be used to carry out a method of using a classifier to assign a label corresponding to a presence, absence, or likelihood of a disease state to data, or to identify the data as indicative or as not indicative of the disease state.
  • the computer system may generate a report identifying a likelihood of the subject having a disease state.
  • the computer system may transmit the report.
  • a diagnostic laboratory may transmit a report regarding the disease state identification to a medical practitioner.
  • a computer system may receive a report.
  • Some aspects include use of a classifier to classify data such as proteomic data obtained from glycoproteins enriched using a method described herein. The classifier may be used to discriminate between the presence of a disease state and a healthy state.
  • a software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.
  • An example storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium.
  • the storage medium may be integral to the processor.
  • the processor and the storage medium may reside in an ASIC.
  • the ASIC may reside in a user terminal.
  • the processor and the storage medium may reside as discrete components in a user terminal.
  • suitable computing devices may include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • server computers desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • Suitable tablet computers may include those with booklet, slate, or convertible configurations, known to those of skill in the art.
  • Described herein are methods that may be useful for disease detection such as cancer detection, or for disease monitoring such as cancer monitoring.
  • a method of the present disclosure may comprise monitoring cancer progression in a patient.
  • Various methods of the present disclosure are able to distinguish between healthy, early stage, and late stage cancers.
  • a method of the present disclosure may also be capable of determining whether a patient is in complete or partial remission.
  • a method may thus comprise analyzing samples from a patient collected at separate points in time. Such methods may identify and then track health or cancer progression in a patient without the need for invasive or expensive procedures. Tracking early phase cancers can be particularly challenging and time intensive for a patient, as small, localized cancers often require biopsies or lengthy imaging sessions for detection. Conversely, the present disclosure provides a variety of methods for tracking small and localized cancers through blood analysis alone.
  • a patient may undergo diagnostic analyses of the present disclosure in daily, twice weekly, weekly, biweekly, monthly, bimonthly, quarterly (once every 3 months), twice yearly, yearly, or biyearly intervals.
  • a patient may be regularly monitored to track remission, early phase cancer status, late phase cancer status, or maintenance of a healthy or pre-cancerous status.
  • the particles and methods of the present disclosure can be used to diagnose cancer up to one year prior, up to two years prior, up to three years prior, up to four years prior, up to five years prior, up to six years prior, up to seven years prior, up to eight years prior, up to nine years prior, up to 10 years prior, up to 15 years prior, up to 20 years prior, or up to 25 years prior to development of symptoms of the cancer.
  • An advantage of many of the methods of the present disclosure may be low invasiveness and minimal patient participation.
  • diagnostic peptides of the present disclosure may be identified in blood (e.g., whole blood, granulocyte, buffy coat, or plasma) samples, and may provide equal or greater diagnostic insight than intensive tissue biopsies or lengthy and expensive imaging procedures.
  • a method described herein may include identifying a biological sample from a subject as being indicative of a healthy state, a cancer state, or a comorbidity thereof in the subject, based on biomarker measurements obtained in the subject.
  • the method may include use of a classifier such as a classifier described herein.
  • the method may distinguish the comorbidity from the cancer state.
  • the method may distinguish the healthy state from the cancer state.
  • the method may distinguish the comorbidity from the healthy state.
  • a method may include comparing an amount of a biomarker to a control.
  • the control may include an index.
  • the control may include a threshold.
  • the control may include a control sample from a control subject. In some cases, the control sample comprises a blood sample, a plasma sample, or a serum sample.
  • Some aspects include obtaining a baseline measurement from the subject. Some aspects include obtaining a baseline biomarker measurement from the subject. Some embodiments include obtaining a measurement from the subject. Some embodiments include obtaining a biomarker measurement from the subject. Some embodiments include comparing the measurement to the baseline measurement. Some embodiments include comparing the biomarker measurement to the baseline biomarker measurement. [0109]
  • the therapeutic intervention may comprise recommending the subject for a secondary clinical test to confirm a diagnosis of the cancer state.
  • This secondary clinical test may comprise an imaging test, a blood test, a computed tomography (CT) scan, a magnetic resonance imaging (MRI) scan, an ultrasound scan, a chest X-ray, a positron emission tomography (PET) scan, a PET-CT scan, a cell-free biological cytology, an amniocentesis, a non-invasive prenatal test (NIPT), or any combination thereof.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • PET-CT scan a cell-free biological cytology
  • amniocentesis a non-invasive prenatal test (NIPT)
  • NIPT non-invasive prenatal test
  • the clinical health data comprises one or more quantitative measures of the subject, such as age, weight, height, body mass index (BMI), blood pressure, heart rate, glucose levels, or a combination thereof.
  • the clinical health data can comprise one or more categorical measures, such as race, ethnicity, history of medication or other clinical treatment, history of tobacco use, history of alcohol consumption, daily activity or fitness level, genetic test results, blood test results, or imaging results.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • a “subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • the term “about” a number refers to that number plus or minus 15% of that number.
  • the term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.
  • treatment or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • a multifunctional molecule represented by Formula 1 (Formula 1), wherein:
  • A is a heterocycle, carbocycle, or trivalent nitrogen
  • L 1 is a linker having the formula: -X ⁇ L ⁇ -CM ⁇ L ⁇ -Y 1 -*, wherein * represents the connection to R 1 ;
  • L 2 is a linker having the formula: -X 2 -L 2a -CM 2 -L 2b -Y 2 -*, wherein * represents the connection to R 2 ;
  • L 3 is a linker having the formula: -X 3 -L 3a -CM 3 -L 3b -Y 3 -*, wherein * represents the connection to R 3 ;
  • CM 1 , CM 2 , and CM 3 are each independently absent or a cleavable moiety, wherein at least one of CM 1 , CM 2 , or CM 3 is a cleavable moiety;
  • L la , L 2a , L 3a , L lb , L 2b , and L 3b are each independently absent or selected from: alkylene and heteroalkylene;
  • X 1 , Y 1 , X 2 , Y 2 , X 3 , and Y 3 are each independently absent or a connecting group;
  • R 1 is a delivery moiety
  • R 2 is a reactive group
  • R 3 is a enrichment handle or is absent.
  • the enrichment handle comprises biotin, desbiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof.
  • the photocleavable moiety comprises a o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o- nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, pivaloyl group, or a benzyl halide.
  • each of the connecting groups of X 1 , Y 1 , X 2 , Y 2 , X 3 , and Y 3 is independently selected from a group consisting of: a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate, 30.
  • each of the connecting groups of X 1 , Y 1 , X 2 , Y 2 , X 3 , and Y 3 is independently selected from a group consisting of: an ester, an amine, an amide, a carbamate, a urea, a thiourea, a triazole, and a carbonate.
  • a method for enrichment of macromolecules comprising: providing a multifunctional molecule comprising: a central moiety, a first arm comprising a delivery moiety connected to the central moiety, and a second arm comprising a reactive group connected to the central moiety; contacting a sample comprising a biological matrix comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule and enriching the macromolecule for further assessment.
  • n comprises 2-8.
  • the biological matrix comprises a biofluid.
  • the biofluid comprises blood, plasma, serum, urine, cerebrospinal fluid, or saliva.
  • cleavable moiety comprises an o-nitrobenzyloxy group, o-nitrobenzylamino group, o- nitrobenzyl group, o-nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, or pivaloyl group.
  • PTM Protein post translational modifications
  • This Example illustrates a multifunctional (trifunctional) molecule that can be modified with different functional groups that allows for the specific capture, enrichment, and subsequent elution of many different types of macromolecule analytes.
  • the multifunctional molecule and method using the multifunctional molecule disclosed herein can be used as a versatile platform to allow a user to profile a specific macromolecule of interest (e.g. glycosylation, phosphorylation, lipids, etc.).
  • a specific macromolecule of interest e.g. glycosylation, phosphorylation, lipids, etc.
  • One advantage is the flexibility it can provide in the specific enrichment and release of a macromolecule of interest after purification.
  • a particular application includes use of a multifunctional molecule comprising a trifunctional molecule is modified with a delivery agent creating the “active” molecule.
  • this delivery agent may bring the entire molecule to the analyte of interest, where the reactive group may introduce a covalent linkage to the analyte.
  • the delivery moiety facilitates the specific linkage of a delivery agent to the trifunctional molecule. Once this delivery agent is linked to the trifunctional molecule, the molecule is active and ready for macromolecule enrichment in biological matrixes.
  • the entire trifunctional molecule-analyte complex may be purified using the purification handle. Once purified, the analyte may then be released into from the trifunctional molecule by utilizing the cleavable linker. Once released, the analyte can be subjected to various analytical characterization techniques.
  • Another example of applying a trifunctional molecule disclosed herein is its ability to enrich glycoproteins from complex matrix.
  • An additional aspect for application of this trifunctional molecule includes global profiling of biological macromolecules that has not been used in a single molecule for specific analyte enrichment.
  • Another aspect of a trifunctional molecule described herein comprises a reactive group.
  • the active molecule can be brought into close proximity macromolecules of interest by the delivery group. Once the analyte of interest is close (distance can vary depending on overall molecular design) to the active trifunctional molecule, the reactive group can introduce a covalent linkage between the trifunctional molecule and the macromolecule.
  • Another aspect of a trifunctional molecule described herein comprises an enrichment handle (or enrichment moiety). After the covalent linkage is introduced to the macromolecule, the entire trifunctional-analyte complex can be captured by affinity enrichment with solid support column. Macromolecules that are not targeted by a delivery agent would not be bound on the support and can be washed away. The targeted analytes can be retained on the support after washing.
  • a trifunctional molecule described herein is a cleavable bond moiety that can be introduced between the central moiety and each of the arms (delivery moiety, reactive group, or enrichment handle). Bond cleavage can be carried out by using UV light, IR light, enzymes, etc. This cleavable bond can be between any combination of arms (FIG. 4-6).
  • the target analytes Once the target analytes are enriched, they can be selectively released from the trifunctional molecule for analysis and characterization. Not only does this allow the user to profile the targeted analytes with multiple workflows, but the released analyte also now harbors a tag that can yield valuable information about the targeted analyte.
  • the trifunctional molecule described herein comprises a central moiety that can have up to three linker arms, each housing a different chemical functionality.
  • FIG. 7 and FIG. 8 illustrate some specific examples of trifunctional molecules that may be used.
  • the counter ion can be trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the trifunctional molecule can be incubated at a temperature between about 4 degrees Celsius to about or about 37 degrees Celsius. In some aspects, the trifunctional molecule can be incubated at 4 degrees Celsius. In some aspects, the trifunctional molecule can be incubated at 37 degrees Celsius.
  • the trifunctional molecule described herein can attach to any azide- modified delivery agent. All copper-free click reactions between an azide and dibenzocyclooctyne (DCBO) can occur at similar reaction conditions, an azide modified delivery agent can be linked to the trifunctional molecule described herein.
  • the length of the PEG (polyethylene glycol)linker can be varied.
  • the PEG linker comprises 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, or more units of PEG.
  • the PEG linker comprises 4 units of PEG and can be decreased or increased as needed to different chemical reactive arm reach with longer PEG arm allowing more promiscuity binding and shorter PEG arms being more specific binding
  • FIG. 7 and FIG. 8 illustrate non-limiting examples of the trifunctional molecules described herein, where the delivery moiety, enrichment moiety, reactive group, and the cleavable bond moiety can be arranged differently for different applications.
  • FIG. 9 illustrates a non-limiting example of a flowchart showing application of a multifunctional molecule described herein for enriching at least one macromolecule from a biological matrix obtained from a subject.
  • Another aspect of a trifunctional molecule described herein comprises a reactive group which can be protected with protecting groups such as Boc (tert-butyloxy carbonyl) and CBz (benzyl chloroformate) protecting groups.
  • protecting groups such as Boc (tert-butyloxy carbonyl) and CBz (benzyl chloroformate) protecting groups.
  • An advantage of protecting groups may be to prevent unwanted side reactions from occurring before delivery of the multifunctional molecule to the desired location.
  • the advantage of the Boc protecting group is that the Boc group will be deprotected under acidic conditions. This means that the trifunctional molecule can be delivered to the glycoprotein of interest, but the covalent linkage between the glycan and multifunctional molecule would not occur until the solution is slightly acifidied to depotect the hydrazide. Then the covalent linkage between the trifunctional molecule and the target glycan can occur. This timing of the reaction may be an advantageous aspect which may lead to less off-target enrichments.
  • the protective group is a Boc protecting group on the reactive group.
  • FIG. 10 provides an example of a Boc protecting group on a hydrazide trifunctional molecule.
  • Concanavalin A was reacted with NHS-PEG4-Azide to introduce azide reactive groups to the N-terminus and lysine residues within the protein.
  • ConA-Azide modified protein was covalently inked to the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule through Cu-free click chemistry between the azide and dibenzylcyclooctyne (DBCO) reactive groups.
  • the N-(DBCO-PEG4)-N-Biotin-PEG4- hydrazide trifunctional molecule was kept at slight molar excess with respect to azide modified ConA protein in a 1.1 : 1 ratio.
  • N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide without ConA was also incubated with the same standard glycoprotein with the same buffers.
  • Each enrichment experiment was performed in triplicate.
  • the stock beads were vortexed briefly and 0.8 mL of the slurry was moved into a new tube. The beads were centrifuged and the supernatant was removed to waste. The beads were washed 3 times with 1 mL of buffer containing 5 mM MgCh 1 mM MnCL and 1 mM CaCh.
  • the beads were resuspended in 0.4 mL of buffer containing 5 mM MgCh 1 mM MnCh and 1 mM CaCh. Following incubation at room temperature for 1 hour with gentle rocking, each sample had 0.05 mL of High-Capacity Streptavidin resin added and was rocked for 30 minutes at room temperature. After incubation, each sample was washed 3 times with 3 M Urea, 5 mM MgCh 1 mM MnCh and 1 mM CaCh. Following washing, the streptavidin beads were pelleted. The pelleted beads were then resuspended in Lyse buffer and protein reduction alkylation, digestion and clean up was performed using PreOmics iST kit following the manufactures guidelines.
  • Peptide samples were then dried by speed vacuum evaporation. All samples were then resuspended with 98%/2% ddH2O/ Acetonitrile with 0.1% Formic Acid. Peptide samples were then analyzed using LC-MS/MS analysis on an Orbitrap Exploris 480 mass spectrometer. The instrument was operated in data dependent acquisition mode. Need to fill in MS details. Data was analyzed using MaxQuant (Version 2.0.3.0) with against human FASTA reviewed database (downloaded 20220806 with 20398 entries) with LFQ quantification enabled.
  • the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide was then exposed to a single oxidized glycoprotein, beta-2-gly coprotein 1, in a buffer, which introduced a covalent modification between the molecule and glycoprotein. Once this covalent modification was introduced, enrichment using an affinity purification column was performed. The column would selectively bind the enrichment moiety on the N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide along with the protein that was covalently linked to it. Wash conditions using high concentrations of protein denaturant were then employed to remove all proteins not covalently bound to the affinity purification column. Remaining proteins were enzymatically digested and analyzed by LC-MS/MS analysis.
  • beta-2-glycoprotein 1 was confidently identified by LCMS in all samples with an average of 9.25 peptides identified per run which equal 32.9% of the entire primary amino acid sequence (FIG. 11).
  • Representative MSI and MS2 of a peptide uniquely originating from the beta-2-gly coprotein 1 can be found in FIG. 12 and FIG. 13.
  • the protein used in this example is for illustrative purposes and other proteins may also be used. Further, the protein may not need to be purified.
  • the second version of the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide molecule investigated was one where the DBCO moiety was reacted with an azide-modified glycoprotein-targeting lectin (Concanavalin A, ConA).
  • ConA glycoprotein-targeting lectin
  • This lectin functions as a delivery agent, bringing the entire enrichment molecule in proximity of the oxidized glycoproteins, where covalent linkage is subsequently introduced.
  • ConA-modified was then exposed to a single oxidized glycoprotein, beta-2-gly coprotein 1, in buffer.
  • the same affinity purification column procedure was employed followed by LC-MS/MS acquisition and data analysis.
  • beta-2-gly coprotein was confidently identified in all replicates with an average of 9.5 unique peptides being identified per run equating to an average sequence coverage of 31.2%.
  • N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide could have a variety of analyte targeting molecules attached to the DBCO moiety without hindering that the hydrazide reactivity with an oxidized glycoprotein (or other aldehyde).
  • N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide molecule Two unique forms of the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide molecule were investigated. The first did not harbor any delivery agent added to the DBCO moiety while the second one had Concanavalin A (ConA), a glycoprotein-binding lectin, attached to the molecule though the DCBO reactive group. First, ConA was reacted with NHS-PEG4-Azide to introduce azide reactive groups to the N-terminus and lysine residues within the protein.
  • ConA Concanavalin A
  • the ConA-azide modified protein was covalently linked to the N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide trifunctional molecule through Cu-free click chemistry between the azide and dibenzylcyclooctyne (DBCO) reactive groups.
  • the N-(DBCO-PEG4)-N-Biotin-PEG4- hydrazide trifunctional molecule was kept at slight molar excess with respect to azide modified ConA protein in a 1.1 : 1 ratio. This ensures all the ConA protein is linked to the N-(DBCO- PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule.
  • terminal sugar moieties on purified 2-gly coprotein 1 standard were oxidized in a solution of 1.5 mM sodium periodate in a buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Both were incubated with the same standard glycoprotein with the same buffers.
  • Each enrichment experiment was performed in triplicate.
  • the stock beads were vortexed briefly and 0.8 mL of the slurry was moved into a new tube. The beads were centrifuged and the supernatant was removed to waste.
  • the beads were washed 3 times with 1 mL of buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. After washing, the beads were resuspended in 0.4 mL of buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Following incubation at room temperature for 1 hour with gentle rocking, each sample had 0.05 mL of High-Capacity Streptavidin resin added and was rocked for 30 minutes at room temperature. After incubation, each sample was washed 3 times with 3 M Urea, 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Following washing, the streptavidin beads were pelleted.
  • the next experiment to be executed is a titration curve experiment with the same beta-2 -glycoprotein 1 used in our initial experiment.
  • the N-(DBCO-PEG4)- N-Biotin-PEG4-hydrazide trifunctional molecule concentration is held constant and the amount of beta-2-gly coprotein 1 is varied over multiple orders of magnitude.
  • protein sample preparation and subsequent digestion and cleanup will be performed as before.
  • the LC-MS/MS analysis and data analysis will be performed on each sample. This experiment will provide insight into capture efficiency of the trifunctional molecule.

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Abstract

Provided are enrichment methods. The methods may be used to enrich macromolecules. The enrichment may be performed with a multifunctional molecule. The multifunctional molecule may include any combination of a delivery moiety, a reactive group, or an enrichment moiety.

Description

MACROMOLECULE ENRICHMENT
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 63/231,616, filed August 10, 2021, and U.S. Provisional Application No. 63/287,209, filed December 8, 2021, which applications are incorporated herein by reference in their entireties.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 59521.709.601. xml, created August 9, 2022, which is 2.03 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
SUMMARY
[0003] Provided herein are multifunctional molecules represented by Formula 1:
Figure imgf000003_0001
(Formula 1), wherein: A is a heterocycle, carbocycle, or trivalent nitrogen; L1 is a linker having the formula: -X^L^-CM^L^-Y1-*, wherein * represents the connection to R':L2 is a linker having the formula: -X2-L2a-CM2-L2b-Y2-*, wherein * represents the connection to R2; L3 is a linker having the formula: -X3-L3a-CM3-L3b-Y3-*, wherein * represents the connection to R3; CM1, CM2, and CM3 are each independently absent or a cleavable moiety, wherein at least one of CM1, CM2, or CM3 is a cleavable moiety; Lla, L2a, L3a, Llb, L2b, and L3b are each independently absent or selected from: alkylene and heteroalkylene; X1, Y1, X2, Y2, X3, and Y3 are each independently absent or a connecting group; R1 is a delivery moiety; R2 is a reactive group; and R3 is a enrichment handle or is absent. In some aspects, the delivery moiety comprises an alkyne. In some aspects, the alkyne comprises a terminal alkyne. In some aspects, the alkyne is a cyclic alkyne. In some aspects, the cyclic alkyne is a DBCO moiety, azacyclooctyne moiety, or cyclooctyne moiety. In some aspects, the cyclic alkyne is a DBCO moiety. In some aspects, the delivery moiety comprises an azide. In some aspects, the reactive moiety comprises a hydrazide. In some aspects, the reactive moiety comprises -C(O)NHNHC(O)OtBu, - C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2. In some aspects, the reactive moiety comprises - C(O)NHNHC(O)OtBu or -C(O)NHNH2. In some aspects, the reactive moiety comprises - C(O)NHNHC(O)OtBu. In some aspects, the reactive moiety comprises -C(O)NHNH2. In some aspects, R3 comprises the enrichment handle, and wherein the enrichment handle comprises biotin, desbiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof. In some aspects, the enrichment handle comprises biotin or desbiotin. In some aspects, the enrichment handle comprises biotin, wherein the cleavable moiety is cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof.
[0004] In some aspects, the cleavable moiety is cleaved by light, an enzyme, or a combination thereof. In some aspects, the light comprises UV light, visible light, IR light, laser, or a combination thereof. In some aspects, the cleavable moiety comprises a photocleavable moiety. In some aspects, the photocleavable moiety comprises a o-nitrobenzyloxy group, o- nitrobenzyl amino group, o-nitrobenzyl group, o-nitroveratryl group, phenacyl group, p- alkoxyphenacyl group, benzoin group, pivaloyl group, or a benzyl halide. In some aspects, the photocleavable moiety comprises the o-nitrobenzyl group. In some aspects, the o-nitrobenzyl group is substituted with a methoxy group or an ethoxy group. In some aspects, the photocleavable moiety is represented by the formula:
Figure imgf000004_0001
[0005] In some aspects, n is selected from any number from 0 to 10. In some aspects, n is 3. In some aspects, the benzyl halide comprises -F, -Cl, -I, -Br, or a combination thereof. In some aspects, each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate, and
Figure imgf000004_0002
[0006] In some aspects, each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: an ester, an amine, an amide, a carbamate, a urea, a thiourea, a triazole, and a carbonate. In some aspects, the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG1-20 or a derivative thereof. In some aspects, the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG4 or a derivative thereof. In some aspects, the alkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a C1-C10 alkylene or a derivative thereof.
[0007] Provided herein are methods for enrichment of macromolecules, comprising: providing a multifunctional molecule comprising: a central moiety, a first arm comprising a delivery moiety connected to the central moiety, and a second arm comprising a reactive group connected to the central moiety; contacting a sample comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule. In some aspects, the method enriches the macromolecule for further assessment. In some aspects, the central moiety comprises a tertiary amine. In some aspects, the delivery moiety is activated with a delivery agent that binds the macromolecule. In some aspects, the delivery agent is azide-modified. In some aspects, the delivery moiety comprises an alkyne. In some aspects, the delivery moiety comprises DBCO moiety, azacyclooctyne moiety or cyclooctyne moiety. In some aspects, the delivery moiety comprises a dibenzocyclooctyne (DBCO) moiety. In some aspects, the first arm comprises a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety. In some aspects, the PEG linker of the first arm comprises PEG1-20. In some aspects, the PEG linker of the first arm comprises PEG2-8. In some aspects, the PEG linker of the first arm comprises PEG4. In some aspects, the second arm comprises a PEG linker connecting the reactive group to the central moiety. In some aspects, the PEG linker of the second arm comprises PEG1-20. In some aspects, the PEG linker of the second arm comprises PEG2-8. In some aspects, the PEG linker of the second arm comprises PEG4. In some aspects, the reactive group comprises a hydrazide reactive group. In some aspects, the reactive group comprises -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2. In some aspects, the reactive group comprises -C(O)NHNH2. In some aspects, the multifunctional molecule further comprises a third arm comprising an enrichment handle connected to the central moiety. In some aspects, the third arm comprises a hydrocarbon connecting the enrichment handle to the central moiety. In some aspects, the enrichment handle comprises biotin or desthiobiotin. In some aspects, the enrichment handle comprises biotin. In some aspects, the multifunctional molecule comprises N-(DBCO-PEGn)-N-Biotin-PEGm-hydrazide or a salt thereof. In some aspects, n comprises 1-20. In some aspects, n comprises 2-8. In some aspects, m comprises 1-20. In some aspects, m comprises 2-8. In some aspects, the multifunctional molecule comprises
Figure imgf000005_0001
some aspects, the multifunctional molecule comprises
Figure imgf000006_0001
. In some aspects, the multifunctional molecule is provided in solution. In some aspects, the multifunctional molecule is provided as a salt with a counterion. In some aspects, the counterion comprises trifluoroacetic acid (TFA). Some aspects include solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule. Some aspects include activating copper-free click chemistry between the delivery moiety and an azide-modified delivery agent prior to contacting the sample with the multifunctional molecule. Some aspects include activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule. In some aspects, activating the click chemistry includes mixing. Some aspects include oxidizing the sample, thereby oxidizing the macromolecule, prior to contacting the sample with the multifunctional molecule. In some aspects, oxidizing the macromolecule generates an aldehyde of the macromolecule, and wherein the aldehyde reacts with the reactive group. Some aspects include binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule. In some aspects, the affinity reagent is connected to a solid support. In some aspects, the affinity reagent comprises avidin or streptavidin. Some aspects include concentrating the captured multifunctional molecule bound to the macromolecule. In some aspects, concentrating comprises precipitating, centrifuging, eluting, or a combination thereof. Some aspects include releasing at least part of the macromolecule from the multifunctional molecule. Some aspects include assaying the macromolecule. In some aspects, the assaying comprises performing mass spectrometry. Some aspects include identifying the macromolecule in the sample as indicative of a biological state. In some aspects, the biological state comprises a disease state. Some aspects include identifying the subject as having the biological state. In some aspects, the macromolecule comprises a protein, carbohydrate, lipid, metabolite or nucleic acid. In some aspects, the protein comprises a post-translational modification. In some aspects, the post- translational modification comprises glycosylation. In some aspects, the protein comprises a glycoprotein or proteoglycan. In some aspects, the sample comprises a biological sample. In some aspects, the biological sample comprises a biological matrix. In some aspects, the biological sample comprises a cell lysate. In some aspects, the biological sample comprises a biofluid sample. In some aspects, the biofluid comprises blood, plasma, serum, urine, cerebrospinal fluid, or saliva. In some aspects, the biological sample is from a subject. In some aspects, the subject is a mammal. In some aspects, the subject is a human. In some aspects, the delivery moiety comprises an antibody. In some aspects, the first arm or the second arm is connected to the central moiety at least in part by a cleavable moiety. In some aspects, the third arm is connected to the central moiety at least in part by a cleavable moiety. In some aspects, the cleavable moiety is photocleavable or enzymatically cleavable. In some aspects, the cleavable moiety comprises an o-nitrobenzyloxy group, o-nitrobenzylamino group, o- nitrobenzyl group, o-nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, or pivaloyl group. In some aspects, the cleavable moiety comprises an o-nitrobenzyl group substituted with a methoxy group or an ethoxy group. In some aspects, the cleavable moiety is represented by the formula:
Figure imgf000007_0001
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 illustrates an example of a multifunctional molecule.
[0009] FIG. 2 illustrates a specific example of a composition comprising a multifunctional molecule.
[0010] FIG. 3 illustrates an example of an enrichment method using a multifunctional molecule.
[0011] FIG. 4 illustrates a non-limiting example of a sketch of a multifunctional molecule described herein, where the multifunctional molecule comprises at least one delivery moiety, at least one react group, a central moiety, and at least one enrichment handle. The at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond. In some cases, the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one uncleavable bond. In some aspects, the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond or at least one uncleavable bond. In some aspects, the at least one delivery moiety, the at least one react group, the central moiety, or the at least one enrichment handle can be connected to each other by at least one cleavable bond and at least one uncleavable bond.
[0012] FIG. 5A-5F illustrate non-limiting examples of the multifunctional molecule described herein showing different arrangements of the cleavable bond.
[0013] FIG. 6 illustrates a non-limiting example of utilizing a multifunctional molecule described herein for an application of analyzing a targeted analyte.
[0014] FIG. 7 illustrates a non-limiting example of a multifunctional molecule described herein, where the reactive group, cleavable bond moiety, enrichment moiety, and the delivery moiety are highlighted.
[0015] FIG. 8 illustrates another non-limiting example of a multifunctional molecule described herein, where the reactive group, cleavable bond moiety, enrichment moiety, and the delivery moiety are highlighted.
[0016] FIG. 9 illustrates a flowchart showing how to utilize a multifunctional molecule described herein for enriching at least one macromolecule from a biological matrix obtained from a subject.
[0017] FIG. 10 illustrates a specific example of a composition comprising a multifunctional molecule and a protecting group on the multifunctional molecule.
[0018] FIG. 11 illustrates SEQ ID NO. 1 with the underlined amino acids designated as peptides found via LC-MS/MS analysis of glycosylated proteins bound to trifunctional molecules.
[0019] FIG. 12 illustrates a mass spectrometry of a protein.
[0020] FIG. 13 illustrates a mass spectrometry of a protein.
DETAILED DESCRIPTION
[0021] Enrichment of macromolecules may be useful for measuring amounts of the macromolecules. For example, proteins with a particular post translational modification (PTM) may be enriched prior to measurement of the proteins.
[0022] Protein glycosylation is an example of a protein PTM that plays a role in numerous biological and disease pathways. Gaining a deeper understanding of global protein glycosylation is useful understanding disease presence and/or progression. A challenge is that protein glycosylation is often difficult to characterize and quantify in many experimental workflows due to their complexity and abundance with respect to other biological compounds. A solution to overcome this difficulty is to enrich glycoproteins or glycopeptides prior to analysis. The methods and compositions such as a multifunctional molecule described herein may be useful for specifically enriching and studying macromolecules such as glycoproteins and glycopeptides. [0023] Disclosed herein are multifunctional molecules. The multifunctional molecule may include a central moiety. The multifunctional molecule may include a first arm comprising a delivery moiety connected to the central moiety, a second arm comprising a reactive group connected to the central moiety, or a third arm comprising an enrichment handle connected to the central moiety, or a combination thereof. In some instances, the first arm, the second arm, or the third arm may comprise a cleavable moiety, such as a photocleavable moiety. The multifunctional molecule may be useful for a method described herein, such as for specifically enriching glycoproteins or other macromolecules from complex biological matrices. Some such molecules may be used to globally profile any or all glycoproteins from a complex biological matrix.
[0024] Also disclosed herein are methods for enrichment of macromolecules. The method may include providing a multifunctional molecule. The method may include contacting a sample comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule. The method may be used to enrich the macromolecules in a biological matrix or fluid.
[0025] A surprising aspect of some embodiments is flexibility of the molecule to attach a delivery agent with ease via a click reaction to the delivery moiety. In some cases, the click reaction may be any copper-free click reactions between an azide and DBCO. In some instances, the delivery agent is azide-modified and the delivery moiety comprises DBCO. In alternative instances, the delivery agent comprises DBCO and the delivery moiety is azide- modified. Any copper-free click reactions between an azide and DBCO, for example, may be performed at similar reaction conditions, so linkage of any azide modified or DBCO modified delivery agent to a multifunctional molecule may be performed.
[0026] Another benefit of some of the methods and compositions described herein is the ability to vary the length of a linker between the central moiety and any one of delivery moiety, reactive group, and enrichment moiety independently. In some cases, the linker comprises an alkylene. In some instances, the alkylene is a C1-C20 alkylene or a derivative thereof. In some instances, the C1-C20 alkylene may optionally be substituted variants thereof. In some instances, the alkylene is a C1-C10 alkylene or a derivative thereof. In some cases, the linker comprises an heteroalkylene. In some instances, the heteroalklyene comprises a PEGi-n, wherein n is any suitable integer. In some instances, n is an integer from 2-100. In some instances, n is an integer from 2-50. In some instances, n is an integer from 2-25. In some instances, n is an integer form 2-20. In some instances, the heteroalkylene comprises a PEGi-20 (e.g. 1 to 2+0.8+0.320 units of polyethene glycol) or a derivative thereof. In some instances, the PEG1-20 may optionally be substituted variants thereof. In some examples, the heteroalkylene comprises a PEG4 or a derivative thereof. In some aspects, the linker may be modified, for example, with a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, or a carbonate. The number of PEG units in a PEG linker or carbon atoms in an alkylene linker can be decreased or increased as needed. Varying the number of PEGs or carbon atoms in the linker may have varying effects chemical reactive arm reach. For example, longer PEG arms may be useful for allowing greater promiscuity, while and shorter PEG arms may provide more specificity.
[0027] A benefit of the compositions and methods herein may be that a macromolecule (e.g. protein or a glycoprotein) may be kept in tact during the enrichment process. As such, a glycosylation site may be identified on the protein or proteins. Some aspects relate to a method of assaying or enriching glycoproteins while keeping the proteins in tact. Some aspects include identifying a site of glycosylation of any of the glycoproteins.
Multifunctional molecules
[0028] Disclosed herein, are multifunctional molecules. An example of a multifunctional molecule is a trifunctional molecule. A multifunctional molecule may include several features. For example, the multifunctional molecule may include multiple features useful for global profiling of glycosylated proteins, which may be used simultaneously in a single molecule for the specific enrichment of glycosylated proteins/peptides. A multifunctional molecule or a trifunctional molecule may include all 3 of the aforementioned features, or a multifunctional molecule may include 1 or 2 of the following features.
• A first feature may include a delivery moiety which can facilitate specific linkage of a delivery agent to the multifunctional molecule using click chemistry. For example, the delivery moiety may comprise a dibenzocyclooctyne (DBCO) moiety and the delivery agent may be azide-modified, which may be linked through copper-free click chemistry. Once the delivery agent is linked to the multifunctional molecule, the molecule is considered active and ready for glycoprotein/gly copeptide enrichment in biological matrixes.
• A second feature may include a reactive group such as a hydrazide reactive group. The active molecule may be brought into close proximity to a glycosylated protein or peptide by the delivery agent. The glycosylated protein or peptide may, for example, be brought into close proximity with the delivery agent via electrostatic interactions. In some examples, the interaction may comprise van der Waals forces. In some examples, the interaction may comprise dipole-dipole interactions. Once in close proximity, the hydrazide group reacts with a previously oxidized sugar (e.g. aldehyde or ketone) on the glycosylated protein or peptide resulting in a covalent linkage between the multifunctional molecule and the glycosylated protein or peptide.
• A third feature of a multifunctional molecule may include an enrichment handle such as a biotin or desthiobiotin enrichment handle. For example, after the covalent linkage is introduced to the sugar, the entire multifunctional molecule-gly coprotein or glycopeptide complex can be captured by affinity enrichment with an avidin or streptavidin solid support. Proteins or peptides that do not have glycosylation sites, may then be washed away after not being bound on the avidin or streptavidin support. The glycosylated proteins or peptides retained on the support after washing may thereby be significantly and specifically enriched, and can then be prepared for analysis assay procedures and.
[0029] Any of the aforementioned features may be connected to a central moiety. The multifunctional molecule may be represented by the formula:
Figure imgf000011_0001
The central moiety may be represented by A. The central moiety may include a heterocycle, a carbocycle, or a trivalent nitrogen. The trivalent nitrogen may include an amine. The amine may include a tertiary amine. Any of the aforementioned features may be connected to the central moiety via a linker (L1, L2, and L3). In such structure, R1 may comprise the delivery moiety, R2 may comprise the reactive group, and R3 may comprise the enrichment handle. The linker may optionally include a cleavable moiety, such as those described herein. The linker may optionally include a connecting group, such as those described herein.
[0030] The delivery moiety may include an alkyne. In some instances, the alkyne is a terminal alkyne. In some instances the alkyne is a cyclic alkyne. The delivery moiety may be ring strained, for example by including a triple bond which is stable when linear. The ring strained delivery moiety may be bent in a non-linear form. The ring strained delivery moiety may be reactive. In some aspects, the delivery moiety is a cyclic alkyne. Cyclic alkyne groups may be useful for the present invention because it allows for azide-modified agents to be attached to the multifunctional group through click chemistry. In some aspects, the delivery moiety is a DBCO moiety. In some aspects, a delivery moiety excludes an amine reactive crosslinker.
[0031] The delivery moiety may be activated with a delivery agent that binds the macromolecule. The delivery agent may be azide-modified. In some cases, the delivery moiety and the delivery agent may react via copper-free click chemistry (e.g., when the delivery moiety comprises DBCO). In some cases, the delivery moiety and the delivery agent may react via copper-catalyzed click chemistry (e.g., when the delivery moiety comprises a terminal alkyne). [0032] A delivery agent may be useful for bringing a macromolecule into proximity with the multifunctional molecule. For example, after a delivery agent reacts and binds covalently with the delivery moiety, the delivery agent covalently bound to the multifunctional molecule may bind non-covalently with the macromolecule. Non-covalent binding of the macromolecule with the activated delivery moiety (comprising the delivery agent bound covalently to the delivery moiety) may bring the macromolecule into proximity with a reactive group of the multifunctional molecule, so that the reactive group may bind covalently to the macromolecule. For example, a multifunctional molecule that includes a delivery moiety comprising DBCO reacted with a delivery agent such as azide-modified affinity reagent (such as a lectin) may be able to bind macromolecules that include sugars. In alternative examples, the functional groups involved in the copper-free click reaction may be interchanged. In such examples, the delivery moiety may be azide-modified and the delivery agent may comprise a DBCO moiety. In some aspects, the delivery agent may comprise an amphiphilic polymer. In some aspects, the delivery agent may comprise a lecithin. In some instances, the lecithin may comprise one or more glycerophospholipids. In some aspects, the one or more glycerophospholipids may comprise phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or phosphatidic acid.
[0033] The first arm may be a linker L1 having the formula: -X^L^-CM^L^-Y1-*, wherein * represents the connection to the delivery moiety. In some instances, CM1 may be a cleavable moiety, such as those described herein. In some instances, CM1 may be absent. In some instances, Lla and Llb may each independently be an alkylene, a heteroalkylene, or an optionally substituted variant thereof, such as those described herein. In some cases, Lla or Llb may each independently is a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety or the cleavable moiety. The PEG linker of the first arm may include PEG2-8. The PEG linker of the first arm may include PEG4. In some instances, Lla and Llb may each independently be absent. In some instances, X1 and Y1, may each independently be a connecting group. In some instances, X1 and Y1, may each independently be absent. In some cases, the connecting group of X1 and Y1 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO2-, - C(O)Ci-C 10 alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-, -C1-C10 alkyl-CO2-, -C1-C10 alkyl-NH-, -C1-C10 alkyl-S-, -C1-C10 alkyl-C(O)-NH-, -C1-C10 alkyl-NH-C(O)-, -CH2CH2S02-Ci-Cio alkyl-, - CH2C(O)-Cl-10 alkyl-, =N-(O orN)-Ci-Cio alkyl-O, =N-(O orN)-Ci-Cio alkyl-NH-, =N-(O or
Figure imgf000013_0001
In some cases, the connecting group of X1 and Y1 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
Figure imgf000013_0002
[0034] The reactive group may include a hydrazide. The hydrazide may react with an oxidized sugar (e.g. aldehyde or ketone) on a glycosylated protein or a glycosylated peptide to form a hydrazone bond between the reactive group and the glycosylated protein or the glycosylated peptide. This may also be lipids or metabolites or other small molecules with an aldehyde functional group.
[0035] The second arm may be a linker L2 having the formula: -X2-L2a-CM2-L2b-Y2-*, wherein * represents the connection to the reactive group. In some instances, CM2 may be a cleavable moiety, such as those described herein. In some instances, CM2 may be absent. In some instances, L2a and L2b may each independently be an alkylene, a heteroalkylene, or a optionally substituted variant thereof, such as those described herein. In some cases, L2a or L2b may each independently is a polyethylene glycol (PEG) linker connecting the reactive group to the central moiety or the cleavable moiety. The PEG linker of the first arm may include PEG2-8. The PEG linker of the first arm may include PEG4. In some instances, L2a and L2b may each independently be absent. In some instances, X2 and Y2, may each independently be a connecting group. In some instances, X2 and Y2, may each independently be absent. In some cases, the connecting group of X2 and Y2 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO2-, - C(O)Ci-C 10 alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-, -C1-C10 alkyl-CO2-, -C1-C10 alkyl-NH-, -C1-C10 alkyl-S-, -C1-C10 alkyl-C(O)-NH-, -C1-C10 alkyl-NH-C(O)-, -CH2CH2S02-Ci-Cio alkyl-, - CH2C(O)-Cl-10 alkyl-, =N-(O orN)-Ci-Cio alkyl-O, =N-(O orN)-Ci-Cio alkyl-NH-, =N-(O or
Figure imgf000014_0001
In some cases, the connecting group of X2 and Y2 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
Figure imgf000014_0002
[0036] The enrichment handle may include biotin, desthiobiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof. The enrichment handle may include biotin. The enrichment handle may include desthiobiotin. The enrichment handle may bind to an affinity reagent. The affinity reagent may be connected to a solid support. The affinity reagent may include avidin. The affinity reagent may include streptavidin. The solid support may include a bead, such as a bead in solution.
[0037] The macromolecule when bound with the multifunctional molecule may be enriched by binding the enrichment handle with any type of solid support, for example, streptavidin coated glass plates to streptavidin coated beads. In such cases, the solid support may be suspended in the sample and then spun down into a pellet bringing the bound macromolecule, while non-enriched macromolecules not bound to multifunctional molecules remain in solution in the sample. Alternatively, the enrichment handle is directly connected to a solid support. [0038] The multifunctional molecule may include a third arm comprising an enrichment handle connected to the central moiety. The second arm may be a linker L3 having the formula: -X3-L3a-CM3-L3b-Y3-*, wherein * represents the connection to the enrichment handle. In some instances, CM3 may be a cleavable moiety, such as those described herein. In some instances, CM3 may be absent. In some instances, L3a and L3b may each independently be an alkylene, a heteroalkylene, or an optionally substituted variant thereof, such as those described herein. In some cases, L3a or L3b may be a C1-C10 alkylene or a derivative thereof. In some cases, L3a or L3b may be absent. In some instances, X3 and Y3, may each independently be a connecting group. In some instances, X3 and Y3, may each independently be absent. In some cases, the connecting group of X3 and Y3 may each independently comprise a -C(O)-, -O-, -S-, - S(O)-, NH-, -C(O)O-, -C(0)Ci-Cio alkyl, -C(0)Ci-Cio alkyl-O-, -C(0)Ci-Cio alkyl-CO2-, - C(0)Ci-Cio alkyl-NH-, -C(0)Ci-Cio alkyl-S-, -C(0)Ci-Cio alkyl-C(O)-NH-, -C(0)Ci-Cio alkyl- NH-C(O)-, -C1-C10 alkyl, -C1-C10 alkyl-O-, -C1-C10 alkyl-CO2-, -C1-C10 alkyl-NH-, -C1-C10 alkyl-S-, -C1-C10 alkyl-C(O)-NH-, -C1-C10 alkyl-NH-C(O)-, -CH2CH2S02-CI-CIO alkyl-, - CH2C(O)-Cl-10 alkyl-, =N-(O orN)-Ci-Cio alkyl-O, =N-(O orN)-Ci-Cio alkyl-NH-, =N-(O or
Figure imgf000015_0001
In some cases, the connecting group of X3 and Y3 may each independently selected from a group consisting of a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
Figure imgf000015_0002
[0039] The linker of any of the arms may include an alkylene. The alkylene may include a Ci-C2o alkylene or a derivative thereof. The alkylene may include a Ci alkylene, a C2 alkylene, a C3 alkylene, a C4 alkylene, a C5 alkylene, a Ce alkylene, a C7 alkylene, a Cx alkylene, a C9 alkylene, a C10 alkylene, a Cn alkylene, a Ci2 alkylene, a C13 alkylene, a C14 alkylene, a C15 alkylene, a Ci6 alkylene, a C17 alkylene, a Cis alkylene, a C19 alkylene, a C2o alkylene, or any range thereof.
[0040] Peptide or peptide-like backbones may be used in a linker or connecting arm. The linker may include an amine - alkyl (e.g. side chain attachment) - carbonyl repeat, or a component thereof. The linker may include an amine. The linker may include an alkyl. The linker may include a carbonyl.
[0041] For example, a biotin may be linked to a central amine directly through a reaction between the amine and biotin. Thus, some aspects do not include a PEG linker in this part of the multifunctional molecule. For synthesizing the multifunctional molecule, a central amine may be reacted with a carboxylic acid of the biotin. In some aspects, the third arm may include a PEG linker connecting the enrichment handle to the central moiety. In aspects that include a PEG linker on the third arm, there may be an alcohol group that forms a bond with biotin. [0042] A trifunctional molecule may include a central tertiary amine that has three linker arms, each housing a different chemical functionality. An example of a multifunctional molecule that includes a trifunctional molecule is N-(DBCO-PEG„)-N-Biotin-PEG„, -hydrazide or a salt thereof. In some aspects, n may be 2-8. For example, n may be 4. In some aspects, m may be 2-8. For example, m may be 4. An example of a multifunctional molecule that includes a trifunctional molecule i
Figure imgf000016_0001
[0043] A trifunctional molecule may include a central tertiary amine that has three linker arms, each housing a different chemical functionality, wherein at least one of the three linkers may comprise a cleavable moiety. In some aspects, at least two of the three linkers may comprise a cleavable moiety. In some aspects, all three of the linkers may comprise a cleavable moiety. In some instances, the cleavable moiety may be between the central moiety and a reactive moiety (e.g., hydrazide), such as in the exemplary formula represented by the structure:
Figure imgf000016_0002
In some instances, the cleavable moiety may between the central moiety and the delivery moiety (e.g., DBCO), such as in the exemplary formula represented by the structure:
Figure imgf000017_0001
[0044] A cleavable moiety may be cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof. In some cases, the light may comprise UV light, visible light, IR light, laser, or a combination thereof. In such cases, the cleavable moiety may be a photocleavable moiety. The photocleaveable moiety may comprise an electron withdrawing group, such as, but not limited to a nitro group or halide group. In alternative cases, the cleavable moiety may be an enzymatically cleavable moiety.
[0045] The cleavable moiety may include a pH sensitive cleavable bond which can be cleaved under acidic or basic conditions. In some non-limiting examples, the cleavable moiety may include a pH sensitive cleavable bond which is cleaved by acidifying the solution. In some non-limiting examples, the cleavable moiety may include a pH sensitive cleavable bond which is cleaved by making the solution more basic. The pH sensitive cleavable bond is advantageous because the molecule can be delivered, but would not react until it was under a slightly acidified environment which can be beneficial for drug delivery.
[0046] The cleavable moiety may include a disulfide bond. The disulfide bond may be chemically or enzymatically formed. The disulfide bond may be cleaved by a reducing agent. The disulfide bond may be enzymatically cleavable. The cleavable moiety may include a protein or peptide sequence that is recognized and cleaved by the enzyme. For example, the cleavable moiety may include the peptide sequence ENLYFQ*S (where * denotes a cleavage site). The disulfide bond may be included as part of a peptide.
[0047] An enzyme that cleaves a cleavable moiety may include an enzyme that cleaves a disulfide bond. Some examples of enzymes that may cleave disulfide bonds include thioredoxin or glutaredoxin. The enzyme may include trypsin. The enzyme may include a virus that cleaves a specific peptide sequence. For example, a tobacco etch virus (TEV) protein that specially cleaves the peptide sequence ENLYFQ*S (where * denotes a cleavage site) may be used. This or another peptide sequence may be present in between the central moiety and one (or any) of the arms. After linkage and enrichment, may bond could be cleaved, thereby releasing the molecule of interest.
[0048] The photocleavable moiety in the trifunctional molecule may be cleaved by UV light. The UV light may have a wavelength in the range of about 100 nm to about 400 nm, about 200 nm to about 400 nm, about 250 nm to about 400 nm, about 280 nm to about 400 nm, about 100 nm to about 370 nm, about 200 nm to about 370 nm, about 250 nm to about 370 nm, or about 280 nm to about 370 nm. In some instances, the photocleavable moiety comprises a nitrobenzyl oxy group, nitrobenzylamino group, nitrobenzyl group, nitroveratryl group, phenacyl group, alkoxyphenacyl group, benzoin group, or a pivaloyl group. In some examples, the nitro group may be in the ortho position of the benzyl, veratryl, phenacyl, benzoin, or pivaloyl group relative to site of cleavage (e.g., o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o-nitroveratryl group). In some examples, the alkoxy group may be in the para position of the benzyl, veratryl, phenacyl, benzoin, or pivaloyl group relative to the site of cleavage (e.g., p-alkoxyphenacyl group). In one aspect, the photocleavable moiety comprises a nitrobenzyl group. The nitro group may be ortho to the benzyl group relative to the site of cleavage (o-nitrobenzyl group). The o-nitrobenzyl group may be substituted with a methoxy or an ethoxy. In some cases, the methoxy or ethoxy may be substituted in the para position relative to the nitro of the o-nitrobenzyl group. In further examples, the o-nitrobenzyl group may comprise a linkage connecting to a linker, such as those described herein, that further connects to the central moiety. The linkage may be in the meta position relative to the nitro group. The linkage may comprise an ester, an ether, an amine, an amide, a carbamate, -O- Ci-Cio alkyl-, or any other linkage described herein. In some examples, the photocleavable moiety may comprise the structure represented by the formula:
Figure imgf000018_0001
such examples, n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, n may be 3.
[0049] The photocleavable moiety in the trifunctional molecule may be cleaved by a laser.
In some cases, the laser is may be in the UV ranges. The UV range may be those described herein. In some instances, the photocleavable moiety comprises a benzyl halide. The halide group may comprise a fluoride, chloride, iodide, bromide, or a combination thereof. In some instances, the photocleavable moiety comprises at least one, at least two, at least three, or at least four halide groups. In some instances, the photocleavable moiety comprises no more than one, no more than two, no more than three, or no more than four halide groups. In some cases, the halide group is a iodide. In such cases, the iodide may be in the ortho position of the benzyl group relative to site of cleavage. In some examples, the benzyl halide may comprise a linkage connected to a linker, such as those described herein, that further connects to the central moiety. The linkage may be in the meta position relative to the halide. The linkage may comprise any linkage structure or connecting structure described herein.
[0050] The multifunctional molecule may include a protecting group. The multifunctional molecule may be deprotected. For example, some aspects include providing a multifunctional molecule including a protecting group, and deprotecting the multifunctional molecule. A reactive moiety of the multifunctional molecule may include the protecting group. An example may include a boc-hydrazide.
[0051] The multifunctional molecule may be included in a composition. The composition may include a solution. The multifunctional molecule may be included in the solution as a salt. The solution may include a counterion such as trifluoroacetic acid (TFA). In some cases, the counter ion may be acetic acid. In some cases, the counter ion may be triethyl ammonium.
Enrichment with multifunctional molecules
[0052] Disclosed herein, in certain aspects, are methods that include enrichment of a macromolecule. The enrichment may be performed using a multifunctional molecule.
[0053] Some aspects include providing a multifunctional molecule. The multifunctional molecule may be provided in solution. An enrichment method may include solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule. The multifunctional molecule may be provided as a salt. The multifunctional molecule may be provided with a counterion, such as those described herein. The counterion may comprise trifluoroacetic acid (TFA). In certain aspects, the counterion does not include hydrochloric acid (HCL). For example, HCL may, in some cases, degrade the multifunctional molecule.
[0054] An enrichment method may include activating click chemistry, such as copper-free or copper-mediated (e.g., Cu(I)) click chemistry. The click chemistry may be between the delivery moiety and a delivery agent. The activation may be before contacting the sample with the multifunctional molecule. The method may include activating copper-free click chemistry between the delivery moiety and the delivery agent prior to contacting the sample with the multifunctional molecule. The delivery moiety may comprise DBCO and the delivery agen may comprise an azide. Alternatively, the delivery moiety may comprise an azide and the delivery agent may comprise a DBCO. Copper free click chemistry between DBCO and an azide group may take place at room temperature with gentle mixing resulting in an “active” molecule. The “active” version of the trifunctional molecule may be mixed at specific ratios with a biological matrix such as plasma, serum, or a cell lysate.
[0055] An azide modified delivery agent and a delivery moiety such as DBCO may be reacted to produce an activated delivery moiety (comprising the delivery agent). Such a reaction may take place by including a mixture of an azide modified delivery agent and DBCO in ethanol and water. This reaction may reach completion after approximately an hour at room temperature with gentle mixing. Mixing may include agitation. Reaction completion can be modified by monitoring absorbance values at 290 nm. This wavelength may be characteristic of DCBO and may disappear or be reduced once the reaction is completed. The active trifunctional molecule may they be mixed with a sample. An incubation time for contacting the biological matrix and trifunctional molecule may be a few hours, and the incubation may be performed at either cold (4 degrees Celsius) or body temperature (37 degrees Celsius). Washing conditions that may be used may be quite stringent since some glycosylated macromolecules may be covalently linked to the trifunctional molecule, and such washing conditions may be useful for specifically enriching glycosylated macromolecules. The enriched macromolecules may then undergo sample preparation steps for assaying the enriched macromolecules.
[0056] A delivery agent may be selected based upon the macromolecule to be enriched. The selected delivery agent may be azide-modified before or as part of a method described herein. The azide modification may enable reaction and binding of the delivery agent to the delivery moiety (for example, to a delivery moiety comprising DBCO).
[0057] A protein can be used as the delivery reagent. For example, the delivery agent may include an antibody. The delivery agent may include any protein or antibody that binds to the macromolecule to be enriched, or a binding fragment thereof. An example of a protein delivery agent includes a lectin protein. The lectin protein may bind glycans. The binding of the lectin protein to a glycan may be specific. The delivery agent may include a commercially available antibody or protein. The antibody may be customized (e.g. non-commercial). Another example of delivery a agent may include an aptamer. The aptamer may be azide modified. The aptamer may be specific to a macromolecule (e.g. protein or type of protein such as glycoprotein). The aptamer may include DNA. The aptamer may include RNA. The aptamer may be modified or include nucleotide modifications. Another example of a delivery agent may include a nanoparticle. The nanoparticle may bind to a macromolecule of interest. The delivery agent may include a protein, antibody, aptamer, or nanoparticle. [0058] The delivery agent may undergo azide introduction to modify the delivery agent (e.g. an antibody or lectin). The azide-modified agent may then be reacted with an alkyne (e.g. terminal or DBCO). The delivery agent may be modified with an azide though routine synthesis, and then attached to the trifunctional molecule and be ready for macromolecule enrichment.
[0059] In an example in which the delivery agent includes an antibody or binding fragment, the antibody may undergo azide introduction to modify the antibody. Similarly, a lectin may be modified with an azide though routine synthesis, then can be attached to the trifunctional molecule and ready for glycan enrichment. Use of an antibody would follow a very similar process in that it would be modified with an azide transfer then would be reacted with the alkyne in a separate second click step.
[0060] The enrichment method may include reacting a delivery agent to the DBCO. The delivery agent may include any type of molecule that brings the multifunctional molecule to a target macromolecule. For example, the delivery agent may include an azide-modified lectin. A lectin may be useful for binding sugars or glycoproteins. The delivery agent may click onto the multifunctional molecule. The multifunctional molecule activated with the delivery agent may be mixed with a sample such as a biological matrix. In the case of the lectin, the lectin may bring the multifunctional molecule to a glycoprotein, where a reactive group (e.g. a hydrazide) of the multifunctional molecule links to the target macromolecule covalently. The glycosylated protein or peptide may, for example, be brought into close proximity with the delivery agent via electrostatic interactions, such as van der Waals forces or dipole-dipole interactions. In some cases, the delivery agent comprises an amphiphilic polymer. The multifunctional molecule linked to the target macromolecule may be enriched, and have non-linked macromolecules washed away.
[0061] As an alternative, copper catalyzed click chemistry may be used. For example, the delivery agent may include an alkyne. The alkyne may be a terminal alkyne.
[0062] Some aspects include activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule. Activating the click chemistry may include gentle mixing.
[0063] Biological samples may be treated with under mild or moderate oxidation conditions, for example, to oxidize either terminal or both terminal and internal sugars. This may result in aldehydes that will react with the hydrazide moiety on our molecule. The method may include oxidizing the sample, thereby oxidizing the macromolecule, prior to contacting the sample with the multifunctional molecule. The method may include oxidizing internal or external sugars of the macromolecule. Oxidizing the macromolecule may generates an aldehyde of the macromolecule. Oxidizing the macromolecule may generates an aldehyde of the internal or external sugars of the glycosylated macromolecule. Examples of oxidizers of a sugar may include, but are not limited to sodium periodate (NalOs), MnO, C2CI2O2, KC Ch, K2CrO4, dess-martin periodinane (CBHBIOS), or perruthenate (e.g., N(C3H?)4RuO4). The aldehyde may react with the reactive group.
[0064] In some aspects, the oxidizer may comprise sodium peroxidate. Terminal glycans can be oxidized under mild concentration of NalCh. Additional, both terminal and internal sugar groups can be oxidized with a higher concentration of NalCh. Examples of mild concentration of NalCh includes, but is not limited to about 1.0 mM of NalCh, 1.5 mM of NalCh, 2.0 mM of NalCE, and 2.5 mM of NalCh. Examples of higher concentration of NalCh includes but is not limited to about 10 mM of NalCh, about 15 mM of NalCh, and about 20 mM of NalCh.
[0065] The method may include binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule. The affinity reagent may be connected to a solid support. The affinity reagent may include avidin. The affinity reagent may include streptavidin.
[0066] The method may include further comprising concentrating the captured multifunctional molecule bound to the macromolecule. For example, the method may include precipitating, centrifuging, eluting, or a combination thereof.
[0067] The macromolecule may then be released and assayed. In some cases, only part of the macromolecule is released or assayed. The method may include releasing at least part of the macromolecule from the multifunctional molecule. The macromolecule may be processed while captured on a solid support such as a bead, and just part of the macromolecule may be released and assayed in some cases.
[0068] The method may include assaying the macromolecule. The assaying may include performing mass spectrometry (MS), chromatography, liquid chromatography (LC), high- performance liquid chromatography, solid-phase chromatography, a lateral flow assay, an immunoassay, an enzyme-linked immunosorbent assay, a western blot, a dot blot, or immunostaining, or a combination thereof. The assaying may include performing MS. MS may be used to assay proteins or other such as metabolites or lipids. For example, the assaying may include LC-MS. Assaying may include use of a protein microarray.
[0069] In some aspects, assaying may include performing nucleic acid sequencing, microarray analysis, hybridization, polymerase chain reaction (PCR), or electrophoresis, or a combination thereof. Assaying may include use of a microarray. Assaying may include use of a nucleic acid microarray. [0070] The assaying may be used to generate assay results. The assay results may include macromolecule measurements. The macromolecule measurements may include protein measurements such as glycoprotein measurements, or a measurement of a protein with a post- translational modification.
[0071] Some aspects include generating the macromolecule measurements. Some aspects include receiving the macromolecule measurements. For example, macromolecule measurements may be received after any of the methods described herein have been performed. [0072] The macromolecule measurements may be useful for identifying a biological state. The method may include identifying the macromolecule in the sample as indicative of a biological state. The biological state may include a disease state. The biological state may include a healthy state. The method may include identifying the subject as having the biological state. In some cases, a healthcare professional receives the macromolecule measurements, or a report based on the macromolecule measurements, and diagnosis the subject as having a disease state or identifies the subject as having a healthy biological state. Some aspects include providing a treatment to the subject based on the disease state. For example, a treatment may include administration of a pharmaceutical composition.
[0073] The method may be used to generate results for multiple macromolecules. For example, the measurements may include measurements of many proteins (e.g. glycoproteins), which may be used in identifying the subject has having the biological state, or in identifying the sample as indicative of the biological state. The measurements may include an omic type of measurement. For example, mass spectrometry may be used to identify all proteins (or other macromolecules) in a sample after enrichment of the sample for the macromolecules. In some aspects, all proteins that have a certain post translational modification such as glycosylation are enriched and measured together using a mass spectrometry or other technique. Then, the measurements may be used as indicated.
[0074] Some aspects relate to a modified or engineered macromolecule such as an engineered protein. The modified or engineered macromolecule may be coupled to a compound described herein. The coupling may include a bond. The bond may be covalent.
Macromolecules
[0075] A variety of macromolecules may be used in the methods described herein. The macromolecule may include a protein, carbohydrate, lipid, metabolite or nucleic acid. The macromolecule may include a protein. The macromolecule may include a carbohydrate. The macromolecule may include a lipid. The macromolecule may include a metabolite. The macromolecule may include a nucleic acid. [0076] The macromolecule may include a protein with a post-translational modification. The post-translational modification may include glycosylation. The protein may include a glycoprotein. The protein may include a proteoglycan. The protein may include a glycosylated protein.
Samples
[0077] A variety of samples may be used in the methods described herein. The sample may include a biological sample. The biological sample may include a biological matrix. The biological sample may include a cell lysate. The biological sample or biological matrix may include a biofluid sample.
[0078] The sample may include a biofluid. The biofluid may include blood, plasma, serum, urine, cerebrospinal fluid, or saliva. The biofluid sample may include a blood sample. The biofluid sample may include a plasma sample. The biofluid sample may include a serum sample. The biofluid sample may include a urine sample. The biofluid sample may include a cerebrospinal fluid sample. The biofluid sample may include a saliva sample. Some examples of biological samples include, but are not limited to, plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, or cell culture samples. In further aspects, the biofluid is plasma or serum. In other aspects, the biofluid is cerebrospinal fluid.
[0079] The subject may be a vertebrate. The subject may be a mammal. The mammal may include a rat, mouse, gerbil, guinea pig, or hamster. The mammal may include a fox, bear, dog, monkey, cow, pig, or sheep. The subject may be a primate. The primate may include an ape or monkey. The primate may include a chimpanzee, a lemur, a bonobo, an orangutan, or a baboon. The subject may be a human. The subject may be an adult (e.g. at least 18-years-old). The subject may be male. The subject may be female. The subject may have a disease state. For example, the subject may have a disease or disorder, a comorbidity of a disease or disorder, or may be healthy.
[0080] A method consistent with the present disclosure may comprise collecting (e.g., isolating, enriching, or purifying) a species from biological sample. The species may be a biomolecule (e.g., a protein), a biomacromolecular structure (e.g., a peptide aggregate or a ribosome), a cell, or tissue. The species may be selectively collected from the biological sample. For example, a method may comprise isolating cancer cells from tissue (e.g., as a tissue biopsy) or from a biofluid (e.g., as a liquid biopsy) such as whole blood, plasma, or a buffy coat. The method may include a sample without cancer cells. The species may be treated prior to analysis. For example, a protein may be reduced and degraded, a nucleic acid may be separated from histones, or a cell may be lysed.
[0081] The biological samples may be obtained or derived from a human subject. The biological samples may be stored in a variety of storage conditions before processing, such as different temperatures (e.g., at room temperature, under refrigeration or freezer conditions, at 25°C, at 4°C, at -18°C, -20°C, or at -80°C) or different suspensions (e.g., EDTA collection tubes).
Diseases
[0082] The methods described herein may be used to evaluate a disease state or a biological state. The methods described herein may be used to predict or identify a biological state. The methods described herein may be used to predict or identify a disease state, or a lack thereof. A treatment may be provided to individuals having a disease state. A disease state may include a disease or disorder such as cancer. Examples of cancer include lung cancer, colon cancer, pancreatic cancer, liver cancer, ovarian cancer, breast cancer, prostate cancer, melanoma, bladder cancer, lymphoma, leukemia, renal cancer, or uterine cancer. In some aspects, the cancer is breast cancer. A disease may include a disorder. A disease state may include having a comorbidity related to a disease or disorder. A reference to whether a subject has a disease state or not may include the subject being healthy. A healthy state may exclude a disease state. For example, a healthy state may exclude having cancer. A disease state may exclude being healthy. [0083] The methods may be useful for cancer diagnosis. The methods may be useful for cancer screening. The method may be useful for cancer treatment. In some cases, the classifier is generated using proteomic data obtained by contacting training samples with particles such that the particles adsorb proteins in the training samples, and assaying the proteins adsorbed to the particles. Some aspects include obtaining of receiving the biofluid sample of the subject.
[0084] The cancer may be at an early stage or a late stage. An example of an early stage of cancer may include stage I. An early stage may include stage I or II. An early stage may include stage I, II, or III. An example of late stage cancer may include stage 4.
Proteomic Data
[0085] The enrichment of macromolecules with the multifunctional molecules described herein may be used to data determination and analysis. The data described herein may include protein data or proteomic data. Proteomic data may involve data about proteins, peptides, or proteoforms. This data may include just peptides or proteins, or a combination of both. An example of a peptide is an amino acid chain. An example of a protein is a peptide or a combination of peptides. For example, a protein may include one, two or more peptides bound together. A protein may be a secreted protein. Proteomic data may include data about various proteoforms. Proteoforms can include different forms of a protein produced from a genome with any variety of sequence variations, splice isoforms, or post-translational modifications. The proteomic data may be generated using an unbiased, non-targeted approach, or may include a specific set of proteins. Aspects described in relation to proteomic data may be relevant to protein data, or vice versa.
[0086] Proteomic data may include information on the presence, absence, or amount of various proteins and peptides. For example, proteomic data may include amounts of proteins. A protein amount may be indicated as a concentration or quantity of proteins, for example a concentration of a protein in a biofluid. A protein amount may be relative to another protein or to another biomolecule. Proteomic data may include information on the presence of proteins or peptides. Proteomic data may include information on the absence of proteins or peptides. Proteomic data may be distinguished by subtype, where each subtype includes a different type of protein, peptide, or proteoform.
[0087] Proteomic data generally includes data on a number of proteins or peptides. For example, proteomic data may include information on the presence, absence, or amount of 1000 or more proteins or peptides. In some cases, proteomic data may include information on the presence, absence, or amount of 5000, 10,000, 20,000, or more peptides, proteins, or proteoforms. Proteomic data may even include up to about 1 million proteoforms. Proteomic data may include a range of proteins, peptides, or proteoforms defined by any of the aforementioned numbers of proteins, peptides, or proteoforms.
[0088] In some aspects, the data comprises measurements of over 10 peptides or protein groups, over 15 peptides or protein groups, over 20 peptides or protein groups, over 25 peptides or protein groups, over 30 peptides or protein groups, over 35 peptides or protein groups, over 40 peptides or protein groups, over 45 peptides or protein groups, over 50 peptides or protein groups, over 75 peptides or protein groups, over 100 peptides or protein groups, over 250 peptides or protein groups, over 500 peptides or protein groups, over 1,000 peptides or protein groups, over 2,500 peptides or protein groups, over 5,000 peptides or protein groups, over 10,000 peptides or protein groups, over 15,000 peptides or protein groups, or over 20,000 peptides or protein groups. In some aspects, the data comprises measurements of at least about 10 peptides or protein groups, at least about 15 peptides or protein groups, at least about 20 peptides or protein groups, at least about 25 peptides or protein groups, at least about 30 peptides or protein groups, at least about 35 peptides or protein groups, at least about 40 peptides or protein groups, at least about 45 peptides or protein groups, at least about 50 peptides or protein groups, at least about 75 peptides or protein groups, at least about 100 peptides or protein groups, at least about 250 peptides or protein groups, at least about 500 peptides or protein groups, at least about 1,000 peptides or protein groups, at least about 2,500 peptides or protein groups, at least about 5,000 peptides or protein groups, at least about 10,000 peptides or protein groups, at least about 15,000 peptides or protein groups, or at least about 20,000 peptides or protein groups. In some aspects, the protein data comprises measurements of no greater than 10 peptides or protein groups, no greater than 15 peptides or protein groups, no greater than 20 peptides or protein groups, no greater than 25 peptides or protein groups, no greater than 30 peptides or protein groups, no greater than 35 peptides or protein groups, no greater than 40 peptides or protein groups, no greater than 45 peptides or protein groups, no greater than 50 peptides or protein groups, no greater than 75 peptides or protein groups, no greater than 100 peptides or protein groups, no greater than 250 peptides or protein groups, no greater than 500 peptides or protein groups, no greater than 1,000 peptides or protein groups, no greater than 2,500 peptides or protein groups, no greater than 5,000 peptides or protein groups, no greater than 10,000 peptides or protein groups, no greater than 15,000 peptides or protein groups, or no greater than 20,000 peptides or protein groups. The peptides or protein groups may comprise or consist of peptides. The peptides or protein groups may comprise or consist of protein groups.
[0089] A protein may also include a post-translational modification (PTM). An example of a PTM may include glycosylation. Proteins or peptides may include glycoproteins or glycopeptides. A protein may include a glycoprotein. A peptide may include a glycopeptide. An example of a PTM includes glycosylation. An example of a PTM may include phosphorylation. Proteins or peptides may include phosphoproteins or phosphopeptides. A protein may include a phosphoprotein. A peptide may include a phosphopeptide.
[0090] Proteomic data may be generated by any of a variety of methods. Generating proteomic data may include using a detection reagent that binds to a peptide or protein and yields a detectable signal. After use of a detection reagent that binds to a peptide or protein and yields a detectable signal, a readout may be obtained that is indicative of the presence, absence or amount of the protein or peptide. Generating proteomic data may include concentrating, filtering, or centrifuging a sample.
[0091] Proteomic data may be generated using mass spectrometry, chromatography, liquid chromatography, high-performance liquid chromatography, solid-phase chromatography, a lateral flow assay, an immunoassay, an enzyme-linked immunosorbent assay, a western blot, a dot blot, or immunostaining, or a combination thereof. Some examples of methods for generating proteomic data include using mass spectrometry, a protein chip, or a reverse-phased protein microarray. Proteomic data may also be generated using an immunoassay such as an enzyme-linked immunosorbent assay, western blot, dot blot, or immunohistochemistry assay. Generating proteomic data may involve use of an immunoassay panel.
[0092] One way of obtaining proteomic data includes use of mass spectrometry. An example of a mass spectrometry method includes use of high resolution, two-dimensional electrophoresis to separate proteins from different samples in parallel, followed by selection or staining of differentially expressed proteins to be identified by mass spectrometry. Another method uses stable isotope tags to differentially label proteins from two different complex mixtures. The proteins within a complex mixture may be labeled isotopically and then digested to yield labeled peptides. Then the labeled mixtures may be combined, and the peptides may be separated by multidimensional liquid chromatography and analyzed by tandem mass spectrometry. A mass spectrometry method may include use of liquid chromatography-mass spectrometry (LC-MS), a technique that may combine physical separation capabilities of liquid chromatography (e.g., HPLC) with mass spectrometry.
[0093] Proteins may be enriched prior to assaying or measuring them. The enrichment may enrich one set of proteins and not another set, or may enrich a single protein and not another protein. Enrichment may be obtained through the use of an affinity reagent, for example by incubating the affinity reagent with a sample prior to measuring proteins in the sample. The affinity reagent may include an antibody. The affinity reagent may include a particle such as a nanoparticle. Proteins may be adsorbed to the affinity reagent, separated from the rest of the sample, and then assayed by using a proteomic assay described herein.
[0094] One aspect of generating proteomic data may include adsorbing proteins (or glycoproteins) to particles such as nanoparticles. Samples may be contacted with particles, for example prior to generating data. The data described herein may generated using particles. For example, a method may include contacting a sample with particles such that the particles adsorb biomolecules (e.g. macromolecules such as proteins). The particles may attract different sets of biomolecules than would normally be measured accurately by performing an omics measurement directly on a sample. For example, a dominant biomolecule may make up a large percentage of certain type of biomolecules (e.g., proteins, transcripts, genetic material, or metabolites) in a sample. For example, one protein may make up a large portion of proteins in circulation that is collected by blood sampling. By adhering biomolecules to particles prior to analyzing the biomolecules, a subset of biomolecules may be obtained that does not include the dominant biomolecule. Removing dominant biomolecules in this way may increase the accuracy of biomolecule measurements and sensitivity of an analysis using those measurements. [0095] Particles can be made from various materials. Such materials may include metals, magnetic particles, polymers, or lipids. A particle may be made from a combination of materials. A particle may comprise layers of different materials. The different materials may have different properties. A particle may include a core comprising one material, and be coated with another material. The core and the coating may have different properties.
[0096] Generating proteomic data may include contacting a sample with particles such that the particles adsorb biomolecules comprising proteins. The adsorbed proteins may be part of a biomolecule corona. The adsorbed proteins may be measured or identified in generating the proteomic data.
[0097] Generating proteomic data may include the use of known amounts internal reference proteins. The reference proteins may be labeled. The label may include an isotopic label.
Generating proteomic data may include the use of known amounts of isotopically labeled, internal reference proteins (referred to as “PiQuant”). The internal reference proteins may be spiked into a sample. The internal reference proteins may be used to identify mass spectra of individual endogenous proteins. The internal reference proteins may be used as standards for determining amounts of the individual endogenous proteins. Proteomic measurements may be generated based on amounts of proteins added into a sample of the one or more biofluid samples. Proteomic measurements may be generated based on amounts of labeled proteins added into a sample of the one or more biofluid samples.
[0098] The protein data or proteomic data may be obtained in conjunction with a method described herein such as an enrichment method. For example, protein data may be obtained after isolation of proteins comprising a particular subgroup such as a PTM using a method disclosed herein. Protein data may be obtained using glycosylated proteins enriched using a method or composition disclosed herein.
Computer systems
[0099] Certain aspects of the methods described herein may be carried out using a computer system. For example, proteomic data analysis may be carried out using a computer system. A readout indicative of the presence, absence or amount of a biomolecule (e.g., proteins such as glycoproteins) may be obtained at least in part using a computer system. The computer system may be used to carry out a method of using a classifier to assign a label corresponding to a presence, absence, or likelihood of a disease state to data, or to identify the data as indicative or as not indicative of the disease state. The computer system may generate a report identifying a likelihood of the subject having a disease state. The computer system may transmit the report. For example, a diagnostic laboratory may transmit a report regarding the disease state identification to a medical practitioner. A computer system may receive a report. Some aspects include use of a classifier to classify data such as proteomic data obtained from glycoproteins enriched using a method described herein. The classifier may be used to discriminate between the presence of a disease state and a healthy state.
[0100] The steps of a method or algorithm described in connection with aspects disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art. An example storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.
[0101] In accordance with the description herein, suitable computing devices may include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers may include those with booklet, slate, or convertible configurations, known to those of skill in the art.
Disease Detection and Monitoring
[0102] Described herein are methods that may be useful for disease detection such as cancer detection, or for disease monitoring such as cancer monitoring.
[0103] A method of the present disclosure may comprise monitoring cancer progression in a patient. Various methods of the present disclosure are able to distinguish between healthy, early stage, and late stage cancers. A method of the present disclosure may also be capable of determining whether a patient is in complete or partial remission. A method may thus comprise analyzing samples from a patient collected at separate points in time. Such methods may identify and then track health or cancer progression in a patient without the need for invasive or expensive procedures. Tracking early phase cancers can be particularly challenging and time intensive for a patient, as small, localized cancers often require biopsies or lengthy imaging sessions for detection. Conversely, the present disclosure provides a variety of methods for tracking small and localized cancers through blood analysis alone. A patient may undergo diagnostic analyses of the present disclosure in daily, twice weekly, weekly, biweekly, monthly, bimonthly, quarterly (once every 3 months), twice yearly, yearly, or biyearly intervals. A patient may be regularly monitored to track remission, early phase cancer status, late phase cancer status, or maintenance of a healthy or pre-cancerous status. In some cases, the particles and methods of the present disclosure can be used to diagnose cancer up to one year prior, up to two years prior, up to three years prior, up to four years prior, up to five years prior, up to six years prior, up to seven years prior, up to eight years prior, up to nine years prior, up to 10 years prior, up to 15 years prior, up to 20 years prior, or up to 25 years prior to development of symptoms of the cancer.
[0104] An advantage of many of the methods of the present disclosure may be low invasiveness and minimal patient participation. In many cases, diagnostic peptides of the present disclosure may be identified in blood (e.g., whole blood, granulocyte, buffy coat, or plasma) samples, and may provide equal or greater diagnostic insight than intensive tissue biopsies or lengthy and expensive imaging procedures.
[0105] A method described herein may include identifying a biological sample from a subject as being indicative of a healthy state, a cancer state, or a comorbidity thereof in the subject, based on biomarker measurements obtained in the subject. The method may include use of a classifier such as a classifier described herein. The method may distinguish the comorbidity from the cancer state. The method may distinguish the healthy state from the cancer state. The method may distinguish the comorbidity from the healthy state.
[0106] Disclosed herein is a method for assaying one or more biomarkers in a sample from a subject suspected of having a cancerous state. The method may include measuring the one or more biomarkers in the sample. The measurement may include detecting a presence of the one or more biomarkers. The measurement may include detecting an absence of the one or more biomarkers. The measurement may include detecting an amount of the one or more biomarkers. [0107] A method may include comparing an amount of a biomarker to a control. The control may include an index. The control may include a threshold. The control may include a control sample from a control subject. In some cases, the control sample comprises a blood sample, a plasma sample, or a serum sample.
[0108] Some aspects include obtaining a baseline measurement from the subject. Some aspects include obtaining a baseline biomarker measurement from the subject. Some embodiments include obtaining a measurement from the subject. Some embodiments include obtaining a biomarker measurement from the subject. Some embodiments include comparing the measurement to the baseline measurement. Some embodiments include comparing the biomarker measurement to the baseline biomarker measurement. [0109] The therapeutic intervention may comprise recommending the subject for a secondary clinical test to confirm a diagnosis of the cancer state. This secondary clinical test may comprise an imaging test, a blood test, a computed tomography (CT) scan, a magnetic resonance imaging (MRI) scan, an ultrasound scan, a chest X-ray, a positron emission tomography (PET) scan, a PET-CT scan, a cell-free biological cytology, an amniocentesis, a non-invasive prenatal test (NIPT), or any combination thereof. The secondary clinical test may comprise a CT scan.
[0110] In some embodiments, for example, the clinical health data comprises one or more quantitative measures of the subject, such as age, weight, height, body mass index (BMI), blood pressure, heart rate, glucose levels, or a combination thereof. As another example, the clinical health data can comprise one or more categorical measures, such as race, ethnicity, history of medication or other clinical treatment, history of tobacco use, history of alcohol consumption, daily activity or fitness level, genetic test results, blood test results, or imaging results.
Definitions
[oni] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0112] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0113] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.
[0114] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
[0115] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease. [0116] As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.
[0117] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
[0118] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
NUMBERED EMBODIMENTS
[0119] Provided herein, are the following embodiments:
1. A multifunctional molecule represented by Formula 1 :
Figure imgf000034_0001
(Formula 1), wherein:
A is a heterocycle, carbocycle, or trivalent nitrogen;
L1 is a linker having the formula: -X^L^-CM^L^-Y1-*, wherein * represents the connection to R1;
L2 is a linker having the formula: -X2-L2a-CM2-L2b-Y2-*, wherein * represents the connection to R2;
L3 is a linker having the formula: -X3-L3a-CM3-L3b-Y3-*, wherein * represents the connection to R3;
CM1, CM2, and CM3 are each independently absent or a cleavable moiety, wherein at least one of CM1, CM2, or CM3 is a cleavable moiety;
Lla, L2a, L3a, Llb, L2b, and L3b are each independently absent or selected from: alkylene and heteroalkylene;
X1, Y1, X2, Y2, X3, and Y3 are each independently absent or a connecting group;
R1 is a delivery moiety;
R2 is a reactive group; and
R3 is a enrichment handle or is absent.
2. The multifunctional molecule of embodiment 1, wherein the delivery moiety comprises an alkyne.
3. The multifunctional molecule of embodiment 2, wherein the alkyne comprises a terminal alkyne.
4. The multifunctional molecule of embodiment 2, wherein the alkyne is a cyclic alkyne.
5. The multifunctional molecule of embodiment 4, wherein the cyclic alkyne is a DBCO moiety, azacyclooctyne moiety, or cyclooctyne moiety.
6. The multifunctional molecule of embodiment 5, wherein the cyclic alkyne is a DBCO moiety.
7. The multifunctional molecule of embodiment 1, wherein the delivery moiety comprises an azide. 8. The multifunctional molecule of any one of embodiments 1-7, wherein the reactive moiety comprises a hydrazide.
9. The multifunctional molecule of embodiment 8, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu, -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2.
10. The multifunctional molecule of embodiment 9, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu or -C(O)NHNH2.
11. The multifunctional molecule of embodiment 10, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu.
12. The multifunctional molecule of embodiment 10, wherein the reactive moiety comprises -C(O)NHNH2.
13. The multifunctional molecule of any one of embodiments 1-12, wherein the enrichment handle comprises biotin, desbiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof.
14. The multifunctional molecule of embodiment 13, wherein the enrichment handle comprises biotin or desbiotin.
15. The multifunctional molecule of embodiment 14, wherein the enrichment handle comprises biotin.
16. The multifunctional molecule of any one of embodiments 1-15, wherein the multifunctional molecule is
Figure imgf000035_0001
17. The multifunctional molecule of any one of embodiments 1- 15, wherein the multifunctional molecule is
Figure imgf000035_0002
18. The multifunctional molecule of any one of embodiments 1-15, wherein the cleavable moiety is cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof. 19. The multifunctional molecule of any one of embodiments 1-15, wherein the cleavable moiety is cleaved by light, an enzyme, or a combination thereof.
20. The multifunctional molecule of embodiment 18, wherein the light comprises UV light, visible light, IR light, laser, or a combination thereof.
21. The multifunctional molecule of any one of embodiments 1-20, wherein the cleavable moiety comprises a photocleavable moiety.
22. The multifunctional molecule of embodiment 21, wherein the photocleavable moiety comprises a o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o- nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, pivaloyl group, or a benzyl halide.
23. The multifunctional molecule of embodiment 22, wherein the photocleavable moiety comprises the o-nitrobenzyl group.
24. The multifunctional molecule of embodiments 22 or 23, wherein the o- nitrobenzyl group is substituted with a methoxy group or an ethoxy group.
25. The multifunctional molecule of any one of embodiments 21-24, wherein the photocleavable moiety is represented by the formula:
Figure imgf000036_0001
26. The multifunctional molecule of embodiment 25, wherein n is selected from any number between 0 to 10.
27. The multifunctional molecule of embodiment 26, wherein n is 3.
28. The multifunctional molecule of embodiment 22, wherein the benzyl halide comprises -F, -Cl, -I, -Br, or a combination thereof.
29. The multifunctional molecule of any one of embodiments 1 to 28, wherein each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
Figure imgf000036_0002
30. The multifunctional molecule of embodiment 29, wherein each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: an ester, an amine, an amide, a carbamate, a urea, a thiourea, a triazole, and a carbonate.
31. The multifunctional molecule of any one of embodiments 1 to 30, wherein the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG1-20 or a derivative thereof.
32. The multifunctional molecule of embodiment 31, wherein the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG4 or a derivative thereof.
33. The multifunctional molecule of any one of embodiments 1-32, wherein the alkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a C1-C10 alkylene or a derivative thereof.
34. A method for enrichment of macromolecules, comprising: providing a multifunctional molecule comprising: a central moiety, a first arm comprising a delivery moiety connected to the central moiety, and a second arm comprising a reactive group connected to the central moiety; contacting a sample comprising a biological matrix comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule and enriching the macromolecule for further assessment.
35. The method of embodiment 34, wherein the central moiety comprises a tertiary amine.
36. The method of embodiment 34 or 35, wherein the delivery moiety is activated with a delivery agent that binds the macromolecule.
37. The method of embodiment 36, wherein the delivery agent is azide-modified.
38. The method of embodiment 36, wherein the delivery moiety comprises an alkyne.
39. The method of embodiment 38, wherein the delivery moiety comprises DBCO moiety, azacyclooctyne moiety or cyclooctyne moiety.
40. The method of embodiment 39, wherein the delivery moiety comprises a dibenzocyclooctyne (DBCO) moiety.
41. The method of any one of embodiments 34-40, wherein the first arm comprises a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety.
42. The method of embodiment 41, wherein the PEG linker of the first arm comprises PEG1-20.
43. The method of embodiment 41 or 42, wherein the PEG linker of the first arm comprises PEG2-8. 44. The method of any one of embodiments 41-43, wherein the PEG linker of the first arm comprises PEG4.
45. The method of any one of embodiments 34-44, wherein the second arm comprises a PEG linker connecting the reactive group to the central moiety.
46. The method of embodiment 45, wherein the PEG linker of the second arm comprises PEG1-20.
47. The method of embodiment 45 or 46, wherein the PEG linker of the second arm comprises PEG2-8.
48. The method of embodiment 45 or 46, wherein the PEG linker of the second arm comprises PEG4.
49. The method of any one of embodiments 34-48, wherein the reactive group comprises a hydrazide reactive group.
50. The method of embodiment 49, wherein the reactive group comprises - C(O)NHNHC(O)OtBu, -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2.
51. The method of embodiment 50, wherein the reactive group comprises - C(O)NHNHC(O)OtBu or -C(O)NHNH2.
52. The method of embodiment 51, wherein the reactive group comprises - C(O)NHNHC(O)OtBu.
53. The method of embodiment 51, wherein the reactive group comprises - C(O)NHNH2.
54. The method of any one of embodiments 34-53, wherein the multifunctional molecule further comprises a third arm comprising an enrichment handle connected to the central moiety.
55. The method of embodiment 54, wherein the third arm comprises a hydrocarbon connecting the enrichment handle to the central moiety.
56. The method of embodiment 54 or 55, wherein the enrichment handle comprises biotin or desthiobiotin.
57. The method of embodiment 56, wherein the enrichment handle comprises biotin.
58. The method of any one of embodiments 34-57, wherein the multifunctional molecule comprises N-(DBCO-PEGn)-N-Biotin-PEGm-hydrazide or a salt thereof.
59. The method of embodiment 58, wherein n comprises 1-20.
60. The method of embodiment 58, wherein n comprises 2-8.
61. The method of embodiment 58, wherein m comprises 1-20.
62. The method of embodiment 58 or 59, wherein m comprises 2-8. 63. The method of any one of embodiments 34-62, wherein the multifunctional molecule comprises
Figure imgf000039_0001
64. The method of any one of embodiments 34-62, wherein the multifunctional molecule comprises
Figure imgf000039_0002
65. The method of any one of embodiments 34-63, wherein the multifunctional molecule is provided in solution.
66. The method of any one of embodiments 34-65, wherein the multifunctional molecule is provided as a salt with a counterion.
67. The method of any one of embodiments 34-66, wherein the counterion comprises trifluoroacetic acid (TFA).
68. The method of any one of embodiments 34-68, further comprising solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule.
69. The method of any one of embodiments 34-36 or 38-68, further comprising activating copper-free click chemistry between the delivery moiety and an azide-modified delivery agent prior to contacting the sample with the multifunctional molecule.
70. The method of any one of embodiments 34-68, further comprising activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule.
71. The method of embodiment 69 or 70, wherein activating the click chemistry comprises mixing.
72. The method of any one of embodiments 34-71, further comprising oxidizing the sample, thereby oxidizing an internal or external sugar of the macromolecule, prior to contacting the sample with the multifunctional molecule. 73. The method of embodiment 72, wherein oxidizing the macromolecule generates an aldehyde of an internal or external sugar of the macromolecule, and wherein the aldehyde reacts with the reactive group.
74. The method of any one of embodiments 34-73, further comprising binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule.
75. The method of embodiment 74, wherein the affinity reagent is connected to a solid support.
76. The method of embodiment 75, wherein the affinity reagent comprises avidin or streptavidin.
77. The method of any one of embodiments 34-76, further comprising concentrating the captured multifunctional molecule bound to the macromolecule.
78. The method of embodiment 77, wherein concentrating comprises precipitating, centrifuging, eluting, or a combination thereof.
79. The method of any one of embodiments 34-78, further comprising releasing at least part of the macromolecule from the multifunctional molecule.
80. The method of any one of embodiments 34-79, further comprising assaying the macromolecule.
81. The method of embodiment 80, wherein the assaying comprises performing mass spectrometry.
82. The method of any one of embodiments 34-81, further comprising identifying the macromolecule in the sample as indicative of a biological state.
83. The method of embodiment 82, wherein the biological state comprises a disease state.
84. The method of any one of embodiments 34-83, wherein the macromolecule comprises a protein, carbohydrate, lipid, metabolite or nucleic acid.
85. The method of embodiment 84, wherein the protein comprises a post- translational modification.
86. The method of embodiment 85, wherein the post-translational modification comprises glycosylation.
87. The method of any one of embodiments 84-86, wherein the protein comprises a glycoprotein or proteoglycan.
88. The method of any one of embodiments 34-87, wherein the biological matrix comprises a biofluid. 89. The method of embodiment 88, wherein the biofluid comprises blood, plasma, serum, urine, cerebrospinal fluid, or saliva.
90. The method of any one of embodiments 34-89, wherein the sample is from a subject.
91. The method of embodiment 90, wherein the subject is a mammal.
92. The method of embodiment 90 or 91, wherein the subject is a human.
93. The method of any one of embodiments 34-92, wherein the delivery moiety comprises an antibody.
94. The method of any one of embodiments 34-93, wherein the first arm or the second arm is connected to the central moiety at least in part by a cleavable moiety.
95. The method of any one of embodiments 34-94, wherein the third arm is connected to the central moiety at least in part by a cleavable moiety.
96. The method of embodiment 94 or 95, wherein the cleavable moiety is photocleavable or enzymatically cleavable.
97. The multifunctional molecule of any one of embodiments 94-96, wherein the cleavable moiety comprises an o-nitrobenzyloxy group, o-nitrobenzylamino group, o- nitrobenzyl group, o-nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, or pivaloyl group.
98. The multifunctional molecule of any one of embodiments 94-97, wherein the cleavable moiety comprises an o-nitrobenzyl group substituted with a methoxy group or an ethoxy group.
99. The multifunctional molecule of any one of embodiments 94-98, wherein the cleavable moiety is represented by the formula:
Figure imgf000041_0001
EXAMPLES
[0120] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way Example 1. Multifunctional and cleavable molecule
[0121] Protein post translational modifications (PTM) may play vital roles in numerous biological and disease pathways. Gaining a deeper understanding of global protein PTMs can be key for reaching a more complete understanding disease state and progression. A challenge encountered in many of these studies is that PTMS may be difficult to characterize and quantify due to their complexity and low abundance levels with respect to other biological components within a sample. A solution to overcome these challenges is to utilize an enrichment prior to analysis.
[0122] This Example illustrates a multifunctional (trifunctional) molecule that can be modified with different functional groups that allows for the specific capture, enrichment, and subsequent elution of many different types of macromolecule analytes. The multifunctional molecule and method using the multifunctional molecule disclosed herein can be used as a versatile platform to allow a user to profile a specific macromolecule of interest (e.g. glycosylation, phosphorylation, lipids, etc.). One advantage is the flexibility it can provide in the specific enrichment and release of a macromolecule of interest after purification.
[0123] A particular application includes use of a multifunctional molecule comprising a trifunctional molecule is modified with a delivery agent creating the “active” molecule. Once active, this delivery agent may bring the entire molecule to the analyte of interest, where the reactive group may introduce a covalent linkage to the analyte. In some aspects, the delivery moiety facilitates the specific linkage of a delivery agent to the trifunctional molecule. Once this delivery agent is linked to the trifunctional molecule, the molecule is active and ready for macromolecule enrichment in biological matrixes.
[0124] Once linked, the entire trifunctional molecule-analyte complex may be purified using the purification handle. Once purified, the analyte may then be released into from the trifunctional molecule by utilizing the cleavable linker. Once released, the analyte can be subjected to various analytical characterization techniques. Another example of applying a trifunctional molecule disclosed herein is its ability to enrich glycoproteins from complex matrix. An additional aspect for application of this trifunctional molecule includes global profiling of biological macromolecules that has not been used in a single molecule for specific analyte enrichment.
[0125] Another aspect of a trifunctional molecule described herein comprises a reactive group. The active molecule can be brought into close proximity macromolecules of interest by the delivery group. Once the analyte of interest is close (distance can vary depending on overall molecular design) to the active trifunctional molecule, the reactive group can introduce a covalent linkage between the trifunctional molecule and the macromolecule. [0126] Another aspect of a trifunctional molecule described herein comprises an enrichment handle (or enrichment moiety). After the covalent linkage is introduced to the macromolecule, the entire trifunctional-analyte complex can be captured by affinity enrichment with solid support column. Macromolecules that are not targeted by a delivery agent would not be bound on the support and can be washed away. The targeted analytes can be retained on the support after washing.
[0127] Another aspect of a trifunctional molecule described herein is a cleavable bond moiety that can be introduced between the central moiety and each of the arms (delivery moiety, reactive group, or enrichment handle). Bond cleavage can be carried out by using UV light, IR light, enzymes, etc. This cleavable bond can be between any combination of arms (FIG. 4-6). Once the target analytes are enriched, they can be selectively released from the trifunctional molecule for analysis and characterization. Not only does this allow the user to profile the targeted analytes with multiple workflows, but the released analyte also now harbors a tag that can yield valuable information about the targeted analyte.
[0128] In some aspects, the trifunctional molecule described herein comprises a central moiety that can have up to three linker arms, each housing a different chemical functionality. FIG. 7 and FIG. 8 illustrate some specific examples of trifunctional molecules that may be used.
[0129] Some specific aspects such as rection conditions may be used. The counter ion can be trifluoroacetic acid (TFA). The trifunctional molecule can be incubated at a temperature between about 4 degrees Celsius to about or about 37 degrees Celsius. In some aspects, the trifunctional molecule can be incubated at 4 degrees Celsius. In some aspects, the trifunctional molecule can be incubated at 37 degrees Celsius.
[0130] In some aspects, the trifunctional molecule described herein can attach to any azide- modified delivery agent. All copper-free click reactions between an azide and dibenzocyclooctyne (DCBO) can occur at similar reaction conditions, an azide modified delivery agent can be linked to the trifunctional molecule described herein. Also, the length of the PEG (polyethylene glycol)linker can be varied. In some aspects, the PEG linker comprises 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, or more units of PEG. In some aspects, the PEG linker comprises 4 units of PEG and can be decreased or increased as needed to different chemical reactive arm reach with longer PEG arm allowing more promiscuity binding and shorter PEG arms being more specific binding
[0131] FIG. 7 and FIG. 8 illustrate non-limiting examples of the trifunctional molecules described herein, where the delivery moiety, enrichment moiety, reactive group, and the cleavable bond moiety can be arranged differently for different applications. FIG. 9 illustrates a non-limiting example of a flowchart showing application of a multifunctional molecule described herein for enriching at least one macromolecule from a biological matrix obtained from a subject.
Example 2. Boc-protected multifunctional molecule
[0132] Another aspect of a trifunctional molecule described herein comprises a reactive group which can be protected with protecting groups such as Boc (tert-butyloxy carbonyl) and CBz (benzyl chloroformate) protecting groups.
[0133] An advantage of protecting groups may be to prevent unwanted side reactions from occurring before delivery of the multifunctional molecule to the desired location. In a nonlimiting example, when the reactive group is a hydrazide, the advantage of the Boc protecting group is that the Boc group will be deprotected under acidic conditions. This means that the trifunctional molecule can be delivered to the glycoprotein of interest, but the covalent linkage between the glycan and multifunctional molecule would not occur until the solution is slightly acifidied to depotect the hydrazide. Then the covalent linkage between the trifunctional molecule and the target glycan can occur. This timing of the reaction may be an advantageous aspect which may lead to less off-target enrichments.
[0134] In some aspects, the protective group is a Boc protecting group on the reactive group. FIG. 10 provides an example of a Boc protecting group on a hydrazide trifunctional molecule.
Example 3. Synthesis of N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide
[0135] Concanavalin A (ConA) was reacted with NHS-PEG4-Azide to introduce azide reactive groups to the N-terminus and lysine residues within the protein. Next, the ConA-Azide modified protein was covalently inked to the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule through Cu-free click chemistry between the azide and dibenzylcyclooctyne (DBCO) reactive groups. The N-(DBCO-PEG4)-N-Biotin-PEG4- hydrazide trifunctional molecule was kept at slight molar excess with respect to azide modified ConA protein in a 1.1 : 1 ratio. This ensures all the ConA protein is linked to the N-(DBCO- PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule. Unreacted N-(DBCO-PEG4)-N- Biotin-PEG4-hydrazide will not negatively impact the subsequent reactions because without the ConA protein, the trifunctional molecule will not be specifically delivered to glycoproteins and purification was omitted. Next, terminal sugar moieties on purified 2-gly coprotein 1 standard (Millipore) was performed in a solution of 1.5 mM sodium periodate in a buffer containing 5 mM MgCh 1 mM MnCh and 1 mM CaCh. As a negative control, N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide without ConA was also incubated with the same standard glycoprotein with the same buffers. Each enrichment experiment was performed in triplicate. To prep the High- Capacity Streptavidin Beads (Thermo) for enrichment, the stock beads were vortexed briefly and 0.8 mL of the slurry was moved into a new tube. The beads were centrifuged and the supernatant was removed to waste. The beads were washed 3 times with 1 mL of buffer containing 5 mM MgCh 1 mM MnCL and 1 mM CaCh. After washing, the beads were resuspended in 0.4 mL of buffer containing 5 mM MgCh 1 mM MnCh and 1 mM CaCh. Following incubation at room temperature for 1 hour with gentle rocking, each sample had 0.05 mL of High-Capacity Streptavidin resin added and was rocked for 30 minutes at room temperature. After incubation, each sample was washed 3 times with 3 M Urea, 5 mM MgCh 1 mM MnCh and 1 mM CaCh. Following washing, the streptavidin beads were pelleted. The pelleted beads were then resuspended in Lyse buffer and protein reduction alkylation, digestion and clean up was performed using PreOmics iST kit following the manufactures guidelines. Peptide samples were then dried by speed vacuum evaporation. All samples were then resuspended with 98%/2% ddH2O/ Acetonitrile with 0.1% Formic Acid. Peptide samples were then analyzed using LC-MS/MS analysis on an Orbitrap Exploris 480 mass spectrometer. The instrument was operated in data dependent acquisition mode. Need to fill in MS details. Data was analyzed using MaxQuant (Version 2.0.3.0) with against human FASTA reviewed database (downloaded 20220806 with 20398 entries) with LFQ quantification enabled.
Example 4. Enrichment Experiments
[0136] Initial experiments examine the feasibility and flexibility of using N-(DBCO- PEG4)-N-Biotin-PEG4-hydrazide as a molecule to introduce covalent linkages to oxidized glycans that can be leveraged for a wide variety of enrichment experiments. With the open flexibly of the molecule, two distinct versions of the molecule were examined. The first of which was nascent N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide where the DCBO moiety was intentionally left unmodified. The N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide was then exposed to a single oxidized glycoprotein, beta-2-gly coprotein 1, in a buffer, which introduced a covalent modification between the molecule and glycoprotein. Once this covalent modification was introduced, enrichment using an affinity purification column was performed. The column would selectively bind the enrichment moiety on the N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide along with the protein that was covalently linked to it. Wash conditions using high concentrations of protein denaturant were then employed to remove all proteins not covalently bound to the affinity purification column. Remaining proteins were enzymatically digested and analyzed by LC-MS/MS analysis. Purified beta-2-glycoprotein 1 was confidently identified by LCMS in all samples with an average of 9.25 peptides identified per run which equal 32.9% of the entire primary amino acid sequence (FIG. 11). Representative MSI and MS2 of a peptide uniquely originating from the beta-2-gly coprotein 1 can be found in FIG. 12 and FIG. 13. The protein used in this example is for illustrative purposes and other proteins may also be used. Further, the protein may not need to be purified.
[0137] The second version of the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide molecule investigated was one where the DBCO moiety was reacted with an azide-modified glycoprotein-targeting lectin (Concanavalin A, ConA). This lectin functions as a delivery agent, bringing the entire enrichment molecule in proximity of the oxidized glycoproteins, where covalent linkage is subsequently introduced. The ConA-modified was then exposed to a single oxidized glycoprotein, beta-2-gly coprotein 1, in buffer. The same affinity purification column procedure was employed followed by LC-MS/MS acquisition and data analysis. Again, it was found that the beta-2-gly coprotein was confidently identified in all replicates with an average of 9.5 unique peptides being identified per run equating to an average sequence coverage of 31.2%. These data indicate that N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide could have a variety of analyte targeting molecules attached to the DBCO moiety without hindering that the hydrazide reactivity with an oxidized glycoprotein (or other aldehyde).
Example 5. Two Forms of the Multifunctional Molecule
[0138] Two unique forms of the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide molecule were investigated. The first did not harbor any delivery agent added to the DBCO moiety while the second one had Concanavalin A (ConA), a glycoprotein-binding lectin, attached to the molecule though the DCBO reactive group. First, ConA was reacted with NHS-PEG4-Azide to introduce azide reactive groups to the N-terminus and lysine residues within the protein. Next, the ConA-azide modified protein was covalently linked to the N-(DBCO-PEG4)-N-Biotin- PEG4-hydrazide trifunctional molecule through Cu-free click chemistry between the azide and dibenzylcyclooctyne (DBCO) reactive groups. The N-(DBCO-PEG4)-N-Biotin-PEG4- hydrazide trifunctional molecule was kept at slight molar excess with respect to azide modified ConA protein in a 1.1 : 1 ratio. This ensures all the ConA protein is linked to the N-(DBCO- PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule. Next, terminal sugar moieties on purified 2-gly coprotein 1 standard were oxidized in a solution of 1.5 mM sodium periodate in a buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Both were incubated with the same standard glycoprotein with the same buffers. Each enrichment experiment was performed in triplicate. To prep the High-Capacity Streptavidin Beads for enrichment, the stock beads were vortexed briefly and 0.8 mL of the slurry was moved into a new tube. The beads were centrifuged and the supernatant was removed to waste. The beads were washed 3 times with 1 mL of buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. After washing, the beads were resuspended in 0.4 mL of buffer containing 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Following incubation at room temperature for 1 hour with gentle rocking, each sample had 0.05 mL of High-Capacity Streptavidin resin added and was rocked for 30 minutes at room temperature. After incubation, each sample was washed 3 times with 3 M Urea, 5 mM MgC12 1 mM MnC12 and 1 mM CaC12. Following washing, the streptavidin beads were pelleted. The proteins bound to the pelleted beads were then digested though standard trypsin digestion methodologies. Peptide samples were then dried by speed vacuum evaporation. All samples were then resuspended with 98%/2% ddH2O/ Acetonitrile with 0.1% Formic Acid. Peptide samples were then analyzed using LC-MS/MS analysis on an Orbitrap Exploris 480 mass spectrometer. The instrument was operated in data dependent acquisition mode. Data was analyzed using MaxQuant (Version 2.0.3.0) with against human FASTA reviewed database (downloaded 20220806 with 20398 entries) with LFQ quantification enabled.
Example 6. Additional Experiments
[0139] The next experiment to be executed is a titration curve experiment with the same beta-2 -glycoprotein 1 used in our initial experiment. In this experiment, the N-(DBCO-PEG4)- N-Biotin-PEG4-hydrazide trifunctional molecule concentration is held constant and the amount of beta-2-gly coprotein 1 is varied over multiple orders of magnitude. After glycoprotein enrichment using the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide reagent, protein sample preparation and subsequent digestion and cleanup will be performed as before. The LC-MS/MS analysis and data analysis will be performed on each sample. This experiment will provide insight into capture efficiency of the trifunctional molecule. After the titration curve experiment out of buffer, a spike in and recovery experiment will be performed. In this experiment, the preparation of the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide trifunctional molecule follows the first experiment. The key difference is our standard glycoprotein will be labeled with heavy isotopes and the enrichment will occur from a complex matrix (e.g. plasma, serum, cell lysate etc.). In the first part of the experiment, a titration curve will be generated by adding various amounts of the heavy isotope-labeled standard glycoprotein into set amounts of complex matrix. After glycoprotein enrichment using the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide reagent, protein sample preparation and subsequent digestion and cleanup will be performed as before. Next, LC-MS/MS analysis and data analysis will be performed on each sample as in the initial experiment, but specific enrichment ratios will be calculated. In the second part of the experiment, the N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide amount added into the complex matrix will be held constant and the amount of complex matrix will be varied. The sample preparation, LC-MS/MS data acquisition and analysis will all be identical as the previous experiments. Specific enrichment ratios again will be calculated. Using these two experiments, optimized amounts of the complex matrix and N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide enrichment molecule will identified. The empirically calculated optimal amounts of each reagent and matrix will be used in the last section of this experiment. No standard protein will be used in this experiment and only endogenous proteins from complex matrix will be examined. The N-(DBCO-PEG4)-N-Biotin-PEG4-hydrazide will be used to identify both new putative glycoproteins and differential abundances of glycoproteins between different samples. [0140] 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. Numerous variations, changes, and substitutions will now 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 in practicing the invention. 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 multifunctional molecule represented by Formula 1 :
Figure imgf000049_0001
(Formula 1), wherein:
A is a heterocycle, carbocycle, or trivalent nitrogen;
L1 is a linker having the formula: -X^L^-CM^L^-Y1-*, wherein * represents the connection to R1;
L2 is a linker having the formula: -X2-L2a-CM2-L2b-Y2-*, wherein * represents the connection to R2;
L3 is a linker having the formula: -X3-L3a-CM3-L3b-Y3-*, wherein * represents the connection to R3;
CM1, CM2, and CM3 are each independently absent or a cleavable moiety, wherein at least one of CM1, CM2, or CM3 is a cleavable moiety;
Lla, L2a, L3a, Llb, L2b, and L3b are each independently absent or selected from: alkylene and heteroalkylene;
X1, Y1, X2, Y2, X3, and Y3 are each independently absent or a connecting group;
R1 is a delivery moiety;
R2 is a reactive group; and
R3 is a enrichment handle or is absent.
2. The multifunctional molecule of claim 1, wherein the delivery moiety comprises an alkyne.
3. The multifunctional molecule of claim 2, wherein the alkyne comprises a terminal alkyne.
4. The multifunctional molecule of claim 2, wherein the alkyne is a cyclic alkyne.
5. The multifunctional molecule of claim 4, wherein the cyclic alkyne is a DBCO moiety, azacyclooctyne moiety, or cyclooctyne moiety.
6. The multifunctional molecule of claim 5, wherein the cyclic alkyne is a DBCO moiety.
7. The multifunctional molecule of claim 1, wherein the delivery moiety comprises an azide.
8. The multifunctional molecule of claim 1, wherein the reactive moiety comprises a hydrazide.
9. The multifunctional molecule of claim 8, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu, -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2.
10. The multifunctional molecule of claim 9, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu or -C(O)NHNH2.
11. The multifunctional molecule of claim 10, wherein the reactive moiety comprises -C(O)NHNHC(O)OtBu.
12. The multifunctional molecule of claim 10, wherein the reactive moiety comprises -C(O)NHNH2.
13. The multifunctional molecule of claim 1, wherein the enrichment handle comprises biotin, desbiotin, an antibody, a protein, a 3x Flag-tag, or a combination thereof.
14. The multifunctional molecule of claim 13, wherein the enrichment handle comprises biotin or desbiotin.
15. The multifunctional molecule of claim 14, wherein the enrichment handle comprises biotin.
16. The multifunctional molecule of claim 1, wherein the multifunctional molecule is
Figure imgf000050_0001
17. The multifunctional molecule of claim 1, wherein the multifunctional molecule is
Figure imgf000050_0002
18. The multifunctional molecule of claim 1, wherein the cleavable moiety is cleaved by light, under acidic conditions, under basic conditions, an enzyme, or a combination thereof.
19. The multifunctional molecule of claim 1, wherein the cleavable moiety is cleaved by light, an enzyme, or a combination thereof.
20. The multifunctional molecule of claim 18, wherein the light comprises UV light, visible light, IR light, laser, or a combination thereof.
21. The multifunctional molecule of claim 1, wherein the cleavable moiety comprises a photocleavable moiety.
22. The multifunctional molecule of claim 21, wherein the photocleavable moiety comprises a o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o- nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, pivaloyl group, or a benzyl halide.
23. The multifunctional molecule of claim 22, wherein the photocleavable moiety comprises the o-nitrobenzyl group.
24. The multifunctional molecule of claim 22, wherein the o-nitrobenzyl group is substituted with a methoxy group or an ethoxy group.
25. The multifunctional molecule of claim 21, wherein the photocleavable moiety is represented by the formula:
Figure imgf000051_0001
26. The multifunctional molecule of claim 25, wherein n is selected from any number between 0 to 10.
27. The multifunctional molecule of claim 26, wherein n is 3.
28. The multifunctional molecule of claim 22, wherein the benzyl halide comprises - F, -Cl, -I, -Br, or a combination thereof.
29. The multifunctional molecule of claim 1, wherein each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: a heterocycle, a carbocycle, an ester, a thioester, an ether, a thioether, an amine, an amide, a carbamate, a urea, a thiourea, a carbonyl, a carbonate,
Figure imgf000051_0002
30. The multifunctional molecule of claim 29, wherein each of the connecting groups of X1, Y1, X2, Y2, X3, and Y3 is independently selected from a group consisting of: an ester, an amine, an amide, a carbamate, a urea, a thiourea, a triazole, and a carbonate.
31. The multifunctional molecule of claim 1, wherein the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG1-20 or a derivative thereof.
32. The multifunctional molecule of claim 31, wherein the heteroalkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a PEG4 or a derivative thereof.
33. The multifunctional molecule of claim 1, wherein the alkylene of Lla, L2a, L3a, Llb, L2b, or L3b is a Ci-C 10 alkylene or a derivative thereof.
34. A method for enrichment of macromolecules, comprising: providing a multifunctional molecule comprising: a central moiety, a first arm comprising a delivery moiety connected to the central moiety, and a second arm comprising a reactive group connected to the central moiety; contacting a sample comprising a biological matrix comprising a macromolecule with the multifunctional molecule, thereby covalently binding the macromolecule to the multifunctional molecule and enriching the macromolecule for further assessment.
35. The method of claim 34, wherein the central moiety comprises a tertiary amine.
36. The method of claim 34, wherein the delivery moiety is activated with a delivery agent that binds the macromolecule.
37. The method of claim 36, wherein the delivery agent is azide-modified.
38. The method of claim 36, wherein the delivery moiety comprises an alkyne.
39. The method of claim 38, wherein the delivery moiety comprises DBCO moiety, azacyclooctyne moiety or cyclooctyne moiety.
40. The method of claim 39, wherein the delivery moiety comprises a dibenzocyclooctyne (DBCO) moiety.
41. The method of claim 34, wherein the first arm comprises a polyethylene glycol (PEG) linker connecting the delivery moiety to the central moiety.
42. The method of claim 41, wherein the PEG linker of the first arm comprises PEG1-20.
43. The method of claim 41 , wherein the PEG linker of the first arm comprises PEG2-8.
44. The method of any claim 1, wherein the PEG linker of the first arm comprises
PEG4.
-50-
45. The method of claim 34, wherein the second arm comprises a PEG linker connecting the reactive group to the central moiety.
46. The method of claim 45, wherein the PEG linker of the second arm comprises PEGI-20.
47. The method of claim 45 , wherein the PEG linker of the second arm comprises PEG2-8.
48. The method of claim 45, wherein the PEG linker of the second arm comprises PEG4.
49. The method of claim 34, wherein the reactive group comprises a hydrazide reactive group.
50. The method of claim 49, wherein the reactive group comprises - C(O)NHNHC(O)OtBu, -C(O)NHNH2, -SO2NHNH2, or -P(O)NHNH2.
51. The method of claim 50, wherein the reactive group comprises - C(O)NHNHC(O)OtBu or -C(O)NHNH2.
52. The method of claim 51, wherein the reactive group comprises - C(O)NHNHC(O)OtBu.
53. The method of claim 51, wherein the reactive group comprises -C(O)NHNH2.
54. The method of claim 34, wherein the multifunctional molecule further comprises a third arm comprising an enrichment handle connected to the central moiety.
55. The method of claim 54, wherein the third arm comprises a hydrocarbon connecting the enrichment handle to the central moiety.
56. The method of claim 54 , wherein the enrichment handle comprises biotin or desthiobiotin.
57. The method of claim 56, wherein the enrichment handle comprises biotin.
58. The method of claim 34, wherein the multifunctional molecule comprises N- (DBCO-PEGn)-N-Biotin-PEGm -hydrazide or a salt thereof.
59. The method of claim 58, wherein n comprises 1-20.
60. The method of claim 58, wherein n comprises 2-8.
61. The method of claim 58, wherein m comprises 1-20.
62. The method of claim 58 , wherein m comprises 2-8.
63. The method of claim 34, wherein the multifunctional molecule comprises
Figure imgf000054_0001
64. The method of claim 34, wherein the multifunctional molecule comprises
Figure imgf000054_0002
65. The method of claim 34, wherein the multifunctional molecule is provided in solution.
66. The method of claim 34, wherein the multifunctional molecule is provided as a salt with a counterion.
67. The method of claim 34, wherein the counterion comprises trifluoroacetic acid (TFA).
68. The method of claim 34, further comprising solubilizing the multifunctional molecule prior to contacting the sample with the multifunctional molecule.
69. The method of claim 34, further comprising activating copper-free click chemistry between the delivery moiety and an azide-modified delivery agent prior to contacting the sample with the multifunctional molecule.
70. The method of claim 34, further comprising activating copper-catalyzed click chemistry between the delivery moiety and a delivery agent prior to contacting the sample with the multifunctional molecule.
71. The method of claim 69, wherein activating the click chemistry comprises mixing.
72. The method of claim 34, further comprising oxidizing the sample, thereby oxidizing an internal or external sugar of the macromolecule, prior to contacting the sample with the multifunctional molecule.
73. The method of claim 72, wherein oxidizing the macromolecule generates an aldehyde of an internal or external sugar of the macromolecule, and wherein the aldehyde reacts with the reactive group.
74. The method of claim 34, further comprising binding the enrichment handle with an affinity reagent, thereby capturing the multifunctional molecule bound to the macromolecule.
75. The method of claim 74, wherein the affinity reagent is connected to a solid support.
76. The method of claim 75, wherein the affinity reagent comprises avidin or streptavidin.
77. The method of claim 34, further comprising concentrating the captured multifunctional molecule bound to the macromolecule.
78. The method of claim 77, wherein concentrating comprises precipitating, centrifuging, eluting, or a combination thereof.
79. The method of claim 34, further comprising releasing at least part of the macromolecule from the multifunctional molecule.
80. The method of claim 34, further comprising assaying the macromolecule.
81. The method of claim 80, wherein the assaying comprises performing mass spectrometry.
82. The method of claim 34, further comprising identifying the macromolecule in the sample as indicative of a biological state.
83. The method of claim 82, wherein the biological state comprises a disease state.
84. The method of claim 34, wherein the macromolecule comprises a protein, carbohydrate, lipid, metabolite or nucleic acid.
85. The method of claim 84, wherein the protein comprises a post-translational modification.
86. The method of claim 85, wherein the post-translational modification comprises glycosylation.
87. The method of claim 84, wherein the protein comprises a glycoprotein or proteoglycan.
88. The method of claim 34, wherein the biological matrix comprises a biofluid.
89. The method of claim 88, wherein the biofluid comprises blood, plasma, serum, urine, cerebrospinal fluid, or saliva.
90. The method of claim 34, wherein the sample is from a subject.
91. The method of claim 90, wherein the subject is a mammal.
92. The method of claim 90 , wherein the subject is a human.
93. The method of claim 34, wherein the delivery moiety comprises an antibody.
-53-
94. The method of claim 34, wherein the first arm or the second arm is connected to the central moiety at least in part by a cleavable moiety.
95. The method of claim 34, wherein the third arm is connected to the central moiety at least in part by a cleavable moiety.
96. The method of claim 94, wherein the cleavable moiety is photocleavable or enzymatically cleavable.
97. The multifunctional molecule of claim 94, wherein the cleavable moiety comprises an o-nitrobenzyloxy group, o-nitrobenzylamino group, o-nitrobenzyl group, o- nitroveratryl group, phenacyl group, p-alkoxyphenacyl group, benzoin group, or pivaloyl group.
98. The multifunctional molecule of claim 94, wherein the cleavable moiety comprises an o-nitrobenzyl group substituted with a methoxy group or an ethoxy group.
99. The multifunctional molecule of claim 94, wherein the cleavable moiety is represented by the formula:
Figure imgf000056_0001
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