WO2021016421A1 - Methods for identifying protein biomarker signatures - Google Patents

Abstract

The present disclosure provides methods for analyzing proteins and protein interactions in a biological sample, including complex samples, such as cells or tissues, comprising of a plurality of proteins. The methods allow detection of a plurality of proteins and protein interactions in a sample.

Description

METHODS FOR IDENTIFYING PROTEIN BIOMARKER SIGNATURES

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Application No. 62/878,282, filed July 24, 2019, which is incorporated herein by reference in its entirety.

BACKGROUND

[002] 1. Field of the Discovery. The present disclosure provides methods for analyzing proteins and protein interactions in a biological sample, including complex samples, such as cells or tissues, comprising of a plurality of proteins. The methods allow detection of a plurality of proteins and protein interactions in a sample.

[003] 2. Background Information. Protein preparations from cells and tissues are extremely complex, with estimated dynamic range (the number of unique species of proteins) in cells believed to be 10⁵-10⁶; and in biological fluids such as serum, potentially greater than 10¹⁰. Currently, there are no parallel protein analysis methods that can interrogate such a large dynamic range. The parallel protein-based analytic methods, two-dimensional electrophoresis (2-DE), and liquid chromatography-mass spectrometry (LC-MS) can access a dynamic range of around 10³ to 10⁵, at best, in an individual experiment. Therefore, only a small portion of the total potential protein complement of a system under analysis is visible to the analyst.

[004] Large proteomic analysis projects aimed at identifying biomarker signatures by interrogating complex protein preparations from equally large numbers of samples requires the ability to view a large number, if not all, proteins in a sample. Current approaches, such as, isoelectric focusing, and 2DE combined with image analysis and the mass spectrometric identification and characterization of digested proteins; subcellular localization, isotope dilution, or shotgun proteomics for capturing cysteinyl peptides by targeting reactive sulfhydryl groups in proteins, phosphopeptides, or glycopeptides, are laborious and time consuming, and also lack the scale, speed, or accuracy needed to yield meaningful correlative data in very large biomarker profiling studies.

[005] Other approaches have been developed for analyzing intact proteins using high- resolution mass spectrometry. Mass spectrometric analysis of protein samples is more easily performed on peptides than intact proteins. Typically, peptides are fragmented in the Mass Spectrometric assay step to yield a multitude of daughter‘ions’ that are then measured and displayed in the resulting MS/MS spectrum. Unfortunately, however, not all peptides are ionized by contemporary approaches, including, electron spray ionization-mass spectrometry (ESI-MS), while some peptides are ionized better by matrix-assisted laser

desorption/ionization-mass spectrometry (MALDI-MS). This has led to incomplete or misleading results, and decreased predictive ability of the assays, only to be overcome with more complex experimental methods employing both LC-MS/MS and on-line fraction collection for subsequent MALDI-MS analysis.

[006] It has been suggested that fractionation of the proteome by specific classes can reduce complexity and increase sensitivity, as well as potentially provide more direct answers to the questions of interest. Clearly, new approaches for profiling proteins or protein fragments from complex biological samples, including cells and tissues, are needed that can interrogate substantial numbers of proteins in the sample.

SUMMARY

[007] Presently described are methods for analyzing proteins in a biological sample, including complex samples, such as cells or tissues, comprising of a plurality of proteins. The methods provided allow characterization of a plurality of proteins in a sample.

Advantageously, the described methods also provide for the reduction in the complexity of the proteins in the sample by apportioning them into a plurality of subsamples. Further, this method is amenable to observing a single protein in the subsample, variants of the single protein, including post-translational forms or isoforms, or the patterns of protein signatures arising from simultaneous observation of a plurality of proteins in the subsample. The peptide signatures generated by a subsample allow characterization of the proteins in the sample as well as a means for differentiating one sample (e.g., cell, tissue, etc.) from another by comparing signatures from various samples. As such, comparisons of signatures between two or more samples can be applied to detect qualitative and quantitative differences of proteins in each of the samples.

[008] Thus, in an aspect, the disclosure provides a method of characterizing a cell or tissue type of interest, the method comprising the steps of: (a) preparing a solid support that comprises a bait protein mixture from a first cell or tissue lysate or extract or fraction from a subject, wherein the bait proteins are immobilized on a surface of the solid support; (b) preparing at least one test cell or tissue lysate sample (i.e., the at least one test sample), wherein the at least one test sample comprises a mixture of two or more prey proteins (i.e., a prey protein mixture); (c) contacting the solid support with the at least one test sample thereby allowing proteins from the bait protein and prey protein mixtures to form protein complexes on the solid support; (d) eluting the bound test sample proteins from the complexes on the solid support; (e) detecting the identity of the eluted proteins from the at least one test sample, thereby obtaining a protein signature for the at least one test sample; and (f) characterizing the cell or tissue type on the basis of the protein signature.

[009] In any aspect or embodiment described herein, the proteins in the bait protein mixture, the at least one test sample or both are at least one of native, partially denatured, fragmented, renatured, mutated, recombinantly or non-recombinantly expressed. In any aspect or embodiment described herein, the proteins in the bait protein mixture, the at least one test sample or both are not exogenously expressed nor recombinant proteins. In any aspect or embodiment described herein, the bait protein mixture, at least one prey protein mixture or both are selected from the group of whole cell protein extract or a fraction thereof.

[0010] In any aspect or embodiment described herein, the at least one test sample comprises from 100 prey proteins to 25000 prey proteins or more. In any aspect or embodiment described herein, the bait protein and prey protein mixtures form a protein complex that comprises three or more proteins.

[0011] In any aspect or embodiment described herein, each bait protein in the bait protein mixture is individually affixed to the solid support.

[0012] In any aspect or embodiment described herein, the at least one test sample is from at least one of the same subject as the first cell or tissue lysate or extract or fraction, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof.

[0013] In additional aspects and embodiments, the method further includes repeating steps (b)-(e) with an additional test sample.

[0014] In any aspect or embodiment described herein, the additional test sample is from at least one of the same subject, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof, as the test sample from step (c).

[0015] In any aspect or embodiment described herein, the additional test sample is from the same cell or tissue type as the at least one test sample from step (c) following treatment with an external stimuli.

[0016] In any aspect or embodiment described herein, the external stimuli is drug treatment, temperature change, irradiation, physical agitation, contact with another cell or tissue, pH change, change in ion concentration, genetic modification of cell, an infected cell or tissue or any treatment which alters proteins or the biology of the cell.

[0017] In any aspect or embodiment described herein, the protein signature is a protein interaction signature.

[0018] In any aspect or embodiment described herein, the methods further comprise the step of profiling the protein signature with known protein interactors on a computer display.

[0019] In any aspect or embodiment described herein, the method comprises, prior to step (d) the step of washing the solid support with buffer solution to remove non-specific binding proteins. In any aspect or embodiment described herein, wherein step (d) includes eluting both the complexed test sample proteins as well as the proteins bound to the solid support.

[0020] In any aspect or embodiment described herein, step (a), step (b) or both includes treating the bait protein mixture, the at least one test sample or both to select or isolate one or more proteins of interest or providing a protein of interest. In any aspect or embodiment described herein, the treatment includes at least one of treatment with a chemical agent, treatment with an affinity reagent, modifying the pH, modifying the ionic concentration, adding or removing divalent cations, modifying the temperature, adding a reducing agent, adding a chaotropic agent, adding a denaturant, adding an additive or an enzyme or a combination thereof.

[0021] In any aspect or embodiment described herein, wherein the bait protein mixture from a first cell or tissue lysate is coupled directly to the surface of the solid support or is immobilized by binding to an affinity matrix on the surface of the solid support.

[0022] In any aspect or embodiment described herein, wherein the step of eluting comprises eluting selectively a population of the bound proteins from the complexes on the solid support.

[0023] In any aspect or embodiment described herein, wherein the bait protein mixture, prey protein mixture or both is from a cancer cell or cancer tissue.

[0024] In any aspect or embodiment described herein, wherein the bait proteins are coupled to the solid support via cross-linking, hydrophobic interactions, an affinity matrix, or combination thereof.

[0025] In any aspect or embodiment described herein, the step of contacting is performed under at least one of the following conditions: low ionic strength, medium ionic strength, high ionic strength, pH < 5, pH 5-8, pH > 8, in the presence of divalent cations, Mg²⁺, Ca²⁺, Mn²⁺, at a temperature < 20 C, at a temperature of 20-22 C, at a temperature of > 22C, or a combination thereof.

[0026] In any aspect or embodiment described herein, the step of eluting selectively comprises eluting proteins using at least one of heat, ionic strength, pH, a chaotropic agent, small molecule inhibitor, allosteric binding agent, competitive binding agent, or a

combination thereof.

[0027] In any aspect or embodiment described herein, the detecting step comprises performing at least one of trypsin digestion or fragmentation of proteins in the eluate, and performing m/z mass spectrometry identification of resulting protein or peptide fragments, amino acid sequencing of protein or peptide fragments, database searching, gel

electrophoresis, detecting with an antibody or a combination thereof.

[0028] In any aspect or embodiment described herein, the method further comprises a step of identifying a parent protein based on the sequence of the peptide fragments, gel mobility, or antibody specificity or a combination thereof.

[0029] In any aspect or embodiment described herein, the methods comprise the step of identifying at least one parent protein in the ubiquitin protein degradation pathway.

[0030] In any aspect or embodiment described herein, the method further comprises the step of characterizing the test sample based on the signature of ubiquitin-protein degradation pathway proteins specific to the test sample.

[0031] In any aspect or embodiment described herein, the ubiquitin signature or protein interaction signature comprises at least one ubiquitin parent protein and at least one protein known or believed to interact with the parent protein.

[0032] In any aspect or embodiment described herein, the method comprises a step subsequent to characterizing the test sample, administering a therapeutic agent to the subject from which the test sample was taken. In any aspect or embodiment described herein, the method comprises changing or modifying at least one of a therapeutic agent, dosage of a therapeutic agent, a dosage regimen or a combination thereof of a subject based on the protein signature.

[0033] In any aspect or embodiment described herein, the solid support is a resin, a membrane, a chip, a cartridge, a size fractionating column, an ion exchange column or matrix, or an affinity matrix.

[0034] In any aspect or embodiment described herein, the method further comprises the steps of preparing an additional test sample, producing an additional protein interaction signature as described herein, e.g, for the ubiquitin-protein degradation pathway proteins in the additional test sample, and comparing the signatures to detect for a change, wherein a change is indicative of a modification of an altered state of the cells. In any aspect or embodiment described herein, the change is indicative of a disease state.

[0035] The preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims.

Additional objects and advantages associated with the compositions, methods, and processes of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional aspects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating an embodiment of the disclosure and are not to be construed as limiting the disclosure. Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0037] Figure 1. Representational diagram of exemplary biological“observational space,” 101.

[0038] Figure 2. Representation of proteins, 102, migrated into the observational space, 101 after treatment of sample or subsample with a condition, hereinafter termed,“lOx.” Proteins 1 03, 104, 105, or 106 in subsample 107 failed to migrate into observational space under condition lOx.

[0039] Figure 3A and Figure 3B. Schematic of Complexity Reduction method - Collecting Proteins entering the observational window and proteins not entering the observational window. Proteins in the observational space 101, are subject to treatment 108, which causes the proteins to migrate into subsample 102 and subsample 107, which comprises proteins 103, 104, 105, and 106.

[0040] Figure 4. Schematic of Complexity Reduction method - Treating Subsample 2 (including subsample 107) under additional conditions 109, 110, 111, and 112, which favor the migration of subsets of proteins (103, 104, 105 and 106) from subsample 107 into an observational window lOla-d, respectively.

[0041] Figure 5. Detection of 62 E3 ligases and their lineage specificities utilizing the methods as described herein. HCC and BCL cell lines show greater congruence with E3 ligase expression compared with BrCa.

[0042] Figure 6. Detection of protein interactions using the methods as described herein.

The methods allow mapping of protein pathways by observing protein-protein interaction networks.

DETAILED DESCRIPTION

[0043] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the disclosure.

[0046] The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0047] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0048] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0049] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0050] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc.

[0051] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0052] The terms "co-administration" and "co-administering" or“combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present compounds described herein, are co-administered in combination with at least one additional bioactive agent, especially including an anticancer agent. In particularly preferred aspects, the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity.

[0053] The term“compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers

(enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context. Deuterated small molecules contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium. [0054] The term“ubiquitin ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin.

[0055] The term“patient” or“subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.

[0056] The term“effective” is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application.

[0057] In a preferred embodiment, this invention describes methods to reduce the complexity of proteins in the sample. Proteins derived from biological materials comprise a complex mixture. Bioessays. 2013 Dec; 35(12): 1050-1055. Published online 2013 Sep 20.

Characterizing how proteins interact, e.g., in a whole cell lysate or cell fraction, can provide valuable information about disease etiology and therapeutic modalities. However, heretofore it has been challenging to assay complex protein interactions in a manner that is reflective of their natural state.

[0058] It is known in the art, that some proteins exist as single polypeptides as determined by their primary amino acid sequence, while others may be comprised of two or more polypeptides held together or somehow associated with one another in their native

environment or during the process of protein preparations from cells and tissues. In addition, the protein or polypeptide may associate with non-protein components, such as nucleic acids, metal ions, membranes, other small or large molecules or other cellular components. Some proteins may exist as multiple forms on account of closely related gene family memberships, alternative splicing, or post- translational modifications. The isoforms could vary in relation to their size, net charge, protein modifications, or other property. According to this invention, and to avoid ambiguity, any protein, polypeptide or isoforms thereof, are described individually or collectively as protein, regardless of which other molecular species it or they may or not be associated with in the native environment, or in the sample of this invention.

[0059] By way of example, phosphorylated proteins possess additional negative charge as compared to the unphosphorylated form of the same protein, and may require a different condition, such as, different pH, ionic concentration, elution buffer compositions, or experimental condition in order to be purified or detected. Likewise, many other kinds of post-translational modifications of proteins exist in biological systems the identification of which can be advantageous in order to characterize in more detail the protein or sample.

Other examples of variability in proteins include proteolytically processing, disulfide bonding, proteins which associate with other proteins in vitro or in vivo to form homo- or hetero-dimers or multimers, protein complexes, positively charged proteins, or membrane- or lipid-associated proteins, and so on.

[0060] It follows that not all species or isoforms of a protein may be recovered using any one condition, and that a plurality of conditions is needed in order to detect many or all types of a protein of interest, isoforms or variants thereof. However, any single condition applied to a biological sample can be expected to yield a plurality of proteins which can be detected by application of available methods. A plurality of conditions applied singly or in combination can greatly improve the chances of characterizing proteins and their variants, as well as the sample in much greater detail.

[0061] Thus, in an aspect, the disclosure provides a method of characterizing a cell or tissue type of interest, the method comprising the steps of: (a) preparing a solid support that comprises a bait protein mixture from a first cell or tissue lysate or extract or fraction from a subject, wherein the bait proteins are immobilized on a surface of the solid support; (b) preparing at least one test cell or tissue lysate sample (i.e., the at least one test sample), wherein the at least one test sample comprises a mixture of two or more prey proteins (i.e., a prey protein mixture); (c) contacting the solid support with the at least one test sample thereby allowing proteins from the bait protein and prey protein mixtures to form protein complexes on the solid support; (d) eluting the bound test sample proteins from the complexes on the solid support; (e) detecting the identity of the eluted proteins from the at least one test sample, thereby obtaining a protein signature for the at least one test sample; and (f) characterizing the cell or tissue type on the basis of the protein signature.

[0062] In any aspect or embodiment described herein, the proteins in the bait protein mixture, the at least one test sample or both are at least one of native, partially denatured, fragmented, renatured, mutated, recombinantly or non-recombinantly expressed. In any aspect or embodiment described herein, the proteins in the bait protein mixture, the at least one test sample or both are not exogenously expressed nor recombinant proteins. In any aspect or embodiment described herein, the bait protein mixture, at least one prey protein mixture or both are selected from the group of whole cell protein extract or a fraction thereof.

[0063] In any aspect or embodiment described herein, the at least one test sample comprises from 100 prey proteins to 25000 prey proteins or more. In any aspect or embodiment described herein, the bait protein and prey protein mixtures form a protein complex that comprises three or more proteins.

[0064] In any of the aspects or embodiments described herein, the test sample comprises from 100 prey proteins or protein complexes to about 25000 or more prey proteins or protein complexes. In any of the aspects or embodiments described herein, the test sample comprises from 500 prey proteins or protein complexes to about 2500 or more prey proteins or protein complexes. In any of the aspects or embodiments described herein, the test sample comprises from 100 prey proteins or protein complexes to about 1000 or more prey proteins or protein complexes. In any of the aspects or embodiments described herein, the test sample comprises from 50 prey proteins or protein complexes to about 200 or more prey proteins or protein complexes. In any of the aspects or embodiments described herein, the test sample comprises from 10 prey proteins or protein complexes to about 100 or more prey proteins or protein complexes In any of the aspects or embodiments described herein, the test sample comprises from 5 prey proteins or protein complexes to about 50 or more prey proteins or protein complexes. In any of the aspects or embodiments described herein, the test sample comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more prey proteins or protein complexes. In certain embodiments, the test sample comprises 2500 or more prey proteins or protein complexes.

[0065] In any of the aspects or embodiments described herein, the bait protein and prey protein mixtures form a protein complex that comprises three or more proteins. In any of the aspects or embodiments described herein, the bait protein and prey protein mixtures form a protein complex that comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 (including values in between) or more proteins In any of the aspects or embodiments described herein, the protein complex comprises from 2-5 proteins, from 4-8 proteins, from 7-12 proteins, or from 12-20 proteins.

[0066] In any aspect or embodiment described herein, each bait protein in the bait protein mixture is individually affixed to the solid support.

[0067] In any aspect or embodiment described herein, the at least one test sample is from at least one of the same subject as the first cell or tissue lysate or extract or fraction, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof. In any of the aspects or embodiments described herein, the therapeutic agent is selected from the group consisting of: JQ1, AZD5153, and I-BET151.

[0068] In additional aspects and embodiments, the method further includes repeating steps (b)-(e) with an additional test sample. In any of the aspects or embodiments described herein, the method further includes repeating steps (b)-(e) with at least one additional test sample, wherein in step (c) at least one prey protein complexing with a bait protein in turn serves as the bait protein for additional protein interactions (i.e., interactions with proteins in the at least one additional test sample). In any of the aspects or embodiments described herein, the protein signature is a protein interaction network, wherein in step (c) at least one prey protein complexing with a bait protein in turn serves as the bait protein for additional protein interactions (i.e., interactions with proteins in the at least one additional test sample). In any of the aspects or embodiments described herein, the network comprises protein interactions in the same or different complexes on the solid surface.

[0069] In any aspect or embodiment described herein, the additional test sample is from at least one of the same subject, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof, as the test sample from step (c).

[0070] In any aspect or embodiment described herein, the additional test sample is from the same cell or tissue type as the at least one test sample from step (c) following treatment with an external stimuli.

[0071] In any aspect or embodiment described herein, the external stimuli is drug treatment, temperature change, irradiation, physical agitation, contact with another cell or tissue, pH change, change in ion concentration, genetic modification of cell, an infected cell or tissue or any treatment which alters proteins or the biology of the cell. [0072] In any of the aspects or embodiments described herein, the cell or tissue is treated with an agent selected from the group consisting of: JQ1, AZD5153, and I-BET151.

[0073] In any aspect or embodiment described herein, the protein signature is a protein interaction signature. In any of the aspects or embodiments described herein, the protein signature comprises an interaction of at least 2 bait proteins with one or more prey proteins, between 3-15 bait proteins with prey proteins, between 12-50 bait proteins with prey proteins, between 45-500 bait proteins with prey proteins, or between 100-1000 bait proteins with prey proteins.

[0074] In any aspect or embodiment described herein, the methods further comprise the step of profiling the protein signature with known protein interactors on a computer display.

[0075] In any aspect or embodiment described herein, the method comprises, prior to step (d) the step of washing the solid support with buffer solution to remove non-specific binding proteins. In any aspect or embodiment described herein, wherein step (d) includes eluting both the complexed test sample proteins as well as the proteins bound to the solid support.

[0076] In any aspect or embodiment described herein, wherein step (a), step (b) or both includes treating the bait protein mixture, the at least one test sample or both to select or isolate one or more proteins of interest or providing a protein of interest. In any aspect or embodiment described herein, the treatment includes at least one of treatment with a chemical agent, treatment with an affinity reagent, modifying the pH, modifying the ionic

concentration, adding or removing divalent cations, modifying the temperature, adding a reducing agent, adding a chaotropic agent, adding a denaturant, adding an additive or an enzyme or a combination thereof.

[0077] In any aspect or embodiment described herein, the bait protein mixture from a first cell or tissue lysate is coupled directly to the surface of the solid support or is immobilized by binding to an affinity matrix on the surface of the solid support. In any of the aspects or embodiments described herein, the binding is via at least one of covalent, non-covalent, electrostatic, ionic, hydrophobic, metal binding, cross-linking, van der Waals interactions or a combination thereof.

[0078] In any aspect or embodiment described herein, wherein the step of eluting comprises eluting selectively a population of the bound proteins from the complexes on the solid support.

[0079] In any aspect or embodiment described herein, wherein the bait protein mixture, prey protein mixture or both is from a cancer cell or cancer tissue. [0080] In any aspect or embodiment described herein, wherein the bait proteins are coupled to the solid support via cross-linking, hydrophobic interactions, an affinity matrix, or combination thereof.

[0081] In any aspect or embodiment described herein, the step of contacting is performed under at least one of the following conditions: low ionic strength, medium ionic strength, high ionic strength, pH < 5, pH 5-8, pH > 8, in the presence of divalent cations, Mg²⁺, Ca²⁺, Mn²⁺, at a temperature < 20 C, at a temperature of 20-22 C, at a temperature of > 22C, or a combination thereof.

[0082] In any aspect or embodiment described herein, the step of eluting selectively comprises eluting proteins using at least one of heat, ionic strength, pH, a chaotropic agent, small molecule inhibitor, allosteric binding agent, competitive binding agent, or a combination thereof.

[0083] In any aspect or embodiment described herein, the detecting step comprises performing at least one of trypsin digestion or fragmentation of proteins in the eluate, and performing m/z mass spectrometry identification of resulting protein or peptide fragments, amino acid sequencing of protein or peptide fragments, database searching, gel

electrophoresis, detecting with an antibody or a combination thereof.

[0084] In any aspect or embodiment described herein, the method further comprises a step of identifying a parent protein based on the sequence of the peptide fragments, gel mobility, or antibody specificity or a combination thereof.

[0085] In any aspect or embodiment described herein, the methods comprise the step of identifying at least one parent protein in the ubiquitin protein degradation pathway.

[0086] In any aspect or embodiment described herein, the method further comprises the step of characterizing the test sample based on the signature of ubiquitin-protein degradation pathway proteins specific to the test sample.

[0087] In any aspect or embodiment described herein, the ubiquitin signature or protein interaction signature comprises at least one ubiquitin parent protein and at least one protein known or believed to interact with the parent protein.

[0088] In any aspect or embodiment described herein, the at least one test sample is characterized on the basis of the presence of from 2 to 1000 distinct proteins, including at least one ubiquitin-protein degradation pathway protein.

[0089] In any aspect or embodiment described herein, the method comprises a step subsequent to characterizing the test sample, administering a therapeutic agent to the subject from which the test sample was taken. In any aspect or embodiment described herein, the method comprises changing or modifying at least one of a therapeutic agent, dosage of a therapeutic agent, a dosage regime or a combination thereof of a subject based on the protein signature.

[0090] In any aspect or embodiment described herein, the solid support is a resin, a membrane, a chip, a cartridge, a size fractionating column, an ion exchange column or matrix, or an affinity matrix.

[0091] In any aspect or embodiment described herein, the method further comprises the steps of preparing an additional test sample, producing an additional protein interaction signature according to the methods described herein for the ubiquitin-protein degradation pathway proteins in the additional test sample, and comparing the signatures to detect for a change, wherein a change is indicative of a modification of an altered state of the cells. In any aspect or embodiment described herein, the change is indicative of a disease state.

[0092] In any of the aspects or embodiments described herein, the ubiquitin-protein degradation pathway parent proteins comprise at least one of an E3 ligase, an E2 Ligase, a polyubiquitin, a USP, a cullin, a SUMO ligase, a deubiquitinase, a ubiquitin binding protein, a SCF protein, a proteasome protein, a peptidase, an F-box protein, or a combination thereof.

[0093] In any of the aspects or embodiments described herein, ubiquitin-protein degradation pathway parent proteins comprise at least one of AMFR, ASB18, ASB6, BRCC3, COP1, CUL4A, CUL4B, DCAF8, DTX3L, DTX4. HECTD1, HERC1, HERC2, JOSD1, LTN1, MARCH8, MINDY4, MYCBP2, PHC3, PPP1R11, RanBP2, RBBP6, RFFL, RNF111, RNF138, RNF168, RNF180, RNF181, RNF187, RNF213, RNF220, RNF25, RNF31,

RNF40, RNF41, RNF6, RNF8, RPS27A, SIAH1, SIAH2, STUB 1, TOPORS, TRAF2, TRAF6, TRIM32, TRIM37, TRIM41, TRIM24, TRIM72, TTC3, UBA2, UBA6, UBE2V2, UBLCP1, UBR4, UBR5, UBR7, UBXN6, UHRF2, UIMC1, UPL5, USP19, USP20, USP25, USP31, USP32, USP4, USP40, USP8, USP9X, MDM2, VHL, cereblon, XIAP or a combination thereof.

[0094] In any of the aspects or embodiments described herein, the at least one test sample is characterized on the basis of the presence of at least two ubiquitin-protein degradation pathway proteins.

[0095] In any of the aspects or embodiments described herein, the at least one test sample is characterized on the basis of the presence of from 3 to 10 ubiquitin-protein degradation pathway proteins. In any of the aspects or embodiments described herein, the at least one test sample is characterized on the basis of the presence of from 11 to 20 ubiquitin-protein degradation pathway proteins.

[0096] In any of the aspects or embodiments described herein, the at least one test sample is characterized on the basis of the presence of from 21 to 50 ubiquitin-protein degradation pathway proteins.

[0097] In any of the aspects or embodiments described herein, the amino acid sequence is shorter than the full-length sequence or comprises a domain in a ubiquitin-protein

degradation pathway protein. In any of the aspects or embodiments described herein, the domain comprises the interaction site of the ubiquitin-protein degradation pathway protein or that of an interacting protein.

[0098] In any of the aspects or embodiments described herein, the first cell or tissue lysate, the at least one test sample, or the additional test sample are from the same or different cell or tissue types.

[0099] In any of the aspects or embodiments described herein, the method comprises changing or modifying at least one of a therapeutic agent, dosage of a therapeutic agent, a dosage regime or a combination thereof of a subject based on the protein signature and characterization of the cell or tissue.

[00100] In any of the aspects or embodiments described herein, the protein mixtures are derived from a cell, tissue, organ or components thereof.

[00101] In any of the aspects or embodiments described herein, the at least one test sample is derived from a human, a bacterium, virus, microbe, a plant or an animal.

[00102] In any of the aspects or embodiments described herein, the at least one test sample cell is derived from a B cell from a human source.

[00103] In any of the aspects or embodiments described herein, the protein interaction signature is indicative of protein interactions in vivo or in vitro.

[00104] In any of the aspects or embodiments described herein, the amino acid sequence is indicative of the parent protein amino acid sequence.

[00105] In any of the aspects or embodiments described herein, first cell or tissue lysate and the at least one test sample are from different physiological states of the same cell type.

[00106] In any of the aspects or embodiments described herein, the cell or tissue lysate is obtained from a member selected from the group consisting of: a drug-treated cell, a gene edited cell, a diseased cell, a drug-resistant cell, a differentiating cell, a proliferating cell, and a combination thereof.

[00107] In any of the aspects or embodiments described herein, the protein signature comprises at least one ubiquitin-protein degradation pathway parent protein. In any of the aspects or embodiments described herein, the ubiquitin-protein degradation pathway parent proteins comprise at least one of USP5, AMFR, OTUB2, OTUB1, CALR, CAND1,

RAD23B, RPN1, UBA1, UBA52, UBE2R2, UBE2V1, USP14, USP15 or a combination thereof.

[00108] In any of the aspects or embodiments described herein, the ubiquitin-protein degradation pathway further comprises at least one two proteins from the group consisting of: USP5, AMFR, OTUB2, OTUB1, CALR, CAND1, RAD23B, RPN1, UBA1, UBA52, UBE2R2, UBE2V1, USP14, USP15, an ACTIN PROTEIN, an ACTIN RELATED

PROTEIN, AFDN, ANNEXIN PROTEIN, API5, ARF3, ARF4, ARF5, ARF6, ARHGEF2, BAG3, BCAP31, BRD4, CALM1, CANX, CASP14, CD36, CDC42, CFL1, CLIC1, CLTC, COPA, COPB1, CPSF6, CSNK1E, CTNNB1, CTNND1, DAD1, DDOST, DDX17,

DDX39A, DDX39B, DDX3X, DDX5, DDX6, DERL1, DHX9, DIRAS2, DRG1, DSTN, DYNC1H1, DYNLL1, DYNLT1, ECPAS, EDF1, EPRS, ERH, ERLIN2, ERP44, EWSR1, EZR, FASN, FBL, FIGNL1, FN1, FYN, , G3BP1, GAN, GAR1, GCN1, GDI1, GSN, GTPBP1, A HISTONE PROTEIN, A HMG PROTEIN, A HNRNP PROTEIN, HRAS, A HSP PROTEIN, ILF2, ILF3, IP07, KIF5B, KIFC3, LSM3, M6PR, MAPK1, MAPRE3, MCCC2, MLF2, MYH14, MY09A, MY09B, NACA, NCL, NDRG1, NDUFB9, NONO, NOP56, NOP58, NUDT21, NUP210, OSTC, PABPC1, PABPC1L2A, PABPC4, PARK7, PCBP1, PDCD6, PDCD6IP, PDIA3, PDIA4, PDIA6, PFDN2, PFN1, PHB, PHB2, PICALM, PIN1, PKP3, PLEC, PLS3, POF1B, PPIA, PPIB, PPME1, PPP1CB, PPP2CA, PPP2CB, PPP2R1A, PROTEASOME PROTEIN, PTBP1, PTK7, PUR, A RAB PROTEIN, RAC1, RACK1, RALA, RALB, RAN, RANGAP1, RAP2B, RBBP4, RBM14, RBMX, A

RIBOSOMAL PROTEIN, RIT2, RNF39, S100A14, S100A7, SEC23A, SEC23B, SEC24C, SEC24D, SEC31A, SET, SF3B1, SF3B2, SF3B3, SFN, SFPQ, SH3GL1, SHMT2, SLC1A5, SLC25A11, SLC25A3, SLC25A5, SLC25A6, SMARCA4, SND1, AN SNRNP PROTEIN, SNU13, SPATA5, SPTAN1, SPTLC1, SRI, SRSF1, SRSF2, SRSF3, SRSF6, SSR3, SSR4, STIP1, STT3A, STX12, SULT2B 1, SUM04, TAGLN2, TALDOl, TCP1, TECR, TF, TFG, TIAL1, TMCOl, TMED10, TMED9, TOPI, TOP2B, TPD52L2, TPT1, TSPO, A TUBULIN PROTEIN, TUFM, TWF1, VAMP2, VAPB, VCP, VDAC1, VDAC2, VIM, VPS26A, VPS29, XPOl, XP07, XPOT, XRCC6, YBX3, YOD1, and combinations thereof.

[00109] In any of the aspects or embodiments described herein, the methods include an additional step of treating the subject or modifying a treatment regimen based on the protein signature. For example, in certain embodiments, the at least one test sample protein signature is compared to a control, wherein a change in the signature is indicative of a disease state or condition. In certain embodiments, the subject is administered an effective amount of a therapeutic agent to treat the disease state or condition. In any of the aspects or embodiments described herein, the therapeutic agent is selected from the group consisting of: JQ1,

AZD5153, and I-BET151.

[00110] Sample:

[00111] The sample useful for practicing this invention comprises of proteins. In preferred embodiments, the sample comprises of a mixture of proteins, such as a cell lysate, a tissue or organ sample, a biopsied sample, or a biological sample. The sample may be derived from any source, including, humans, animals, plants, microorganisms or may be virus- infected samples. Generally, biological sources are complex mixtures of proteins and other components, and exhibit a variety of structural, chemical, conformational, and functional diversities amongst the proteins therein. Characteristics of the proteins in a sample of this invention include, a diversity of molecular sizes (kDa), net positive or negative charge or uncharged, may exist in one or more conformational states, possess any of a number of chemical modifications, such as, phosphorylation, disulfide bonding (intrachain or interchain), methylation, acetylation, carbo-di-imidylation, ubiquitinylation, glycosylation, or be alternatively spliced forms, isoforms or proteolytically processed forms. Some proteins may interact with other components, including proteins (protein-protein interactions), nucleic acids (protein-nucleic acid interaction) or other molecules, such as, cofactors, ligands, metals, ions, drugs or metabolites or other small or large molecules. Other proteins may associate non-covalently with other proteins or polypeptides via charge or hydrophobic interactions, or be chemically linked via a disulfide or other chemical bonds. Yet other proteins may be conjugated in cells and tissues with other proteins or polypeptides, such as, ubiquitin, or components of the cellular machinery, including structural components. This methods described herein are amenable to the analysis of any of such proteins. Samples may be of biological origin, such as cells, tissues, organs or whole organisms, and may include exogenously introduced proteins, such as via gene transfer or recombinant expression. [00112] In certain embodiments, the sample comprises a mixture of proteins, including, optionally, non-protein material, such as, nucleic acids, carbohydrates, lipids or other small or large molecules. This methods described herein are also amenable to the analysis of proteins in samples that contain non-protein components, regardless of whether the protein or proteins in sample is/are associated with those components. In certain embodiments, the sample comprises a cell or tissue lysate, serum, plasma or a biological fluid.

[00113] Subsample:

[00114] In an additional aspect, this disclosure provides conditions to apportion some of the sample proteins under each condition into a selected pool, termed a subsample. For example, a condition is one or a set of parameters applied to a sample or subsample. In general, and without limitation, a parameter is selected in a manner as to alter the form, structure, conformation, or other characteristic of a protein or proteins in the sample or subsample. In general, a parameter of this invention alters at least one characteristic of a subset of proteins in the sample or subsample so as to permit collection of those proteins in to a different subsample. In one embodiment, sample proteins are separated based on the net charge of the proteins. In an exemplary manner, and without limitation, proteins in the sample can be contacted with a non-protein material, such as a solid surface, support, or matrix, including but not limited to, silica gel, a resin, a membrane, or a bead, that facilitates binding of or association of at least some of the sample proteins to the supporting material. Upon washing, the proteins associated with the material are eluted, so as to prepare a subsample. Ion separation columns are commercially available, such as from Thermo Scientific [Catalog Nos.: 90008, 90010, 89870]. Such columns aid the association of either positively or negatively charged proteins, or hydrophobic proteins. According to the methods described herein, the sample proteins can be applied individually, sequentially, or in combination with any of these columns, bind and elute proteins with selected properties from a sample so as to create subsamples.

[00115] In alternate embodiments, proteins in a sample or subsample can be contacted with a protein or other molecule (such as a drug or metabolite) of interest, and the sample proteins associating with the protein or molecule of interest can be collected into a different subsample. Available methods, such as anchoring the protein of interest, differential centrifugation, affinity capturing can be effectively applied to aid the separation of proteins into the subsample. The associated proteins may also be cross-linked following association using commonly available cross-linking agents, prior to separation from the non-associating proteins or other components in the sample.

[00116] In another aspect, sample proteins are separated by taking advantage of the range of sizes of proteins, such as by passage through a size fractionation column or gel electrophoresis. In certain embodiments, the methods include fractionating proteins in the sample by treating with a size fractionation column. Such columns each generally contain a filter characterized by a pore size (nanometers), which acts as a separation barrier, and under appropriate conditions (such as by application of a g-force, known as centrifugal force), allows proteins smaller than the pore size to pass through and be collected in the flow through or eluate, while retaining larger proteins in the supernatant. Such columns are commercially available, such as from, Thermo Scientific [Pierce Concentrator, PES, 3K,

10K, 30K and 100K MWCO; 0.5ml, Cat. Nos. 88512, 88513, 88502, 88503, respectively] which can be employed for separation of sample proteins in the 3 kDa, 10 kDa, 30 kDa, or 100 kDa size ranges, away from larger proteins in the sample. The conditions for separation may involve denaturing the protein (in presence of a detergent such as SDS), non-denaturing conditions, reducing conditions (in presence of DTT, DMSO) or chaotropic agents

(Guanidine HCL, Betaine, etc.). Likewise, gel filtration columns can be used which exclude proteins small enough to enter the pores of the gel matrix, and let larger proteins to pass through and be collected in the eluate. In another embodiment, separation methods based on protein charge can also be applied in combination with size-based separation methods to provide additional conditions for preparing subsamples. In this manner, the methods of this invention create a plurality of subsamples for detection in subsequent steps.

[00117] In yet another aspect, a condition promoting dissociation of proteins is applied to the sample or subsample of this invention. Without limitation, examples of dissociation of proteins according to this invention applies to the process of separating individual proteins involved in protein complexes, polypeptide chains of any protein comprised of two or more subunits, including proteins involving a disulfide linkage or other covalent modifications necessary to stably hold two or more polypeptide chains together under experimental conditions, a protein associated with at least one other protein, or any that alters the form of structure of the protein.

[00118] In yet another aspect, dissociation of proteins according to this invention also relates to separation of two or more regions of a single polypeptide chain under normal or altered experimental conditions. For instance, applying extreme conditions such as pH, ionic strength, or temperatures to a sample of proteins can disrupt some polypeptide chains or regions thereof or fragment them. In general, applications of such conditions may fragment some polypeptide chains containing certain labile amino acids, modified amino acids or other chemical modifications. For example, treatment of Lysozyme at very high temperatures showed a variety of effects. Nonpolar aliphatic, acidic and aromatic amino acids were all relatively stable with less than 20% disintegration after 24 h at 180 degrees C. Other amino acids were labile, with lability increased in the order proline, arginine, histidine, cysteine, threonine, lysine, tryptophan, serine, and methionine. Methionine was 86% decomposed after 24 h at 180° C. Of the atypical amino acids (lysinoalanine, allo-isoleucine and ornithine), only lysinoalanine was useful as an indicator to detect amino acid damage after dry heating. [Ref: Z Lebensm Unters Forsch. 1983 ; 176(6):421-5] . This invention is amenable to creating subsamples by treating proteins under such conditions which promote fragmentation of polypeptides, including enzymatic fragmentation.

[00119] In yet another aspect, this invention describes the creation of multiple subsamples of proteins starting with proteins in the sample. In one embodiment, a first subsample is created by treating sample proteins with a column of a selected pore size, and a second subsample of predominantly larger proteins retained in the supernatant of the column. The second subsample is subjected to a second condition to prepare two additional subsamples, one responsive to the second condition (third subsample), and another not responsive to the second condition (fourth subsample). Likewise, many other subsamples are created by applying additional conditions. In a subsequent step of this invention, the proteins in any subsample are analyzed. The number of subsamples created in this manner is not important, it can be any that provides an adequate level of analysis to characterize the sample or a protein of interest. As needed, most or all of the proteins in the sample can be‘binned’ or apportioned into one or other subsample of this invention. This invention is amenable to such approaches.

[00120] According to the methods described herein, a protein in the sample can be observed in its native form as present in the sample or recovered in the cell lysate, or can be modified along with or prior to applying one or more steps of this invention, such as, treating sample or subsample with an enzyme, including a protease, reacting with an antibody or affinity molecule, including a small molecule; staining with an affinity reagent which provides a colorimetric observation of one or more proteins in the sample; or by treatments which can remove existing modifications on any protein in the sample. [00121] Accordingly, in any of the aspects or embodiments described herein, the methods include a subsample; a subsample comprises one or a set of proteins in the original sample. In one embodiment, the subsample is prepared by separating the set of proteins substantially away from the remainder of the components in the sample. Methods in the art describe separating proteins based on their size, charge, hydrophobicity, association with a matrix, including a chemically modified substrate, such as an antibody, a small molecule, a metal ion, or a gel. Any of these methods can be used to prepare a subsample.

[00122] In certain embodiments, the subsample comprises of a plurality of proteins or polypeptides, based on a property, such as, size of the protein or range of sizes, net charge, hydrophobicity, association with other proteins or molecules, affinity to associate with a matrix, or stability under the experimental condition or when combined with molecule of interest. It is also known in the art that the net charge on a protein or polypeptide or the ability to associate with other proteins or molecules can vary with the conditions of the experiment, including, but not limited to, salt concentration, pH, temperature, or presence of metal ions or other molecules. Many other approaches have been described in the art to separate a set of proteins away from other components in the sample, including, ammonium sulfate or trichloroacetic acid (TCA) precipitation, or solubilizing proteins in the presence of Guanidinium Hydrochloride. The methods as described herein is amenable to employing any of these conditions while preparing a subsample, individually or sequentially.

[00123] In additional embodiments, this invention describes methods for preparing a plurality of subsamples starting with the sample. Generally, each subsample comprises a set of proteins in the sample, which bear a property characteristic of proteins. For example, a first subsample prepared by the methods of this invention comprises of subjecting sample to a first conditions so as to collect proteins substantially at or below a selected molecular size or range of molecular weights of the proteins. Generally, and without limitation, the first condition is selected in a manner that some, but not all proteins in the sample can be collected in the first subsample. The remaining proteins in the sample after the first condition can then be subjected to a second condition, such as a second, higher molecular weight cut-off, charge, protein association, or other property of proteins, so as to prepare a second subsample. By sequentially selecting conditions for preparation of subsamples, this invention favors apportioning many or all of the proteins in the sample into a plurality of subsamples, thereby reducing the complexity of the subsample as it relates to the original complexity. [00124] The methods as described herein are also amenable to analyzing only a subset, but not all, proteins in the sample. Therefore, in certain embodiments, the methods include analyzing a subset but not all of the proteins from a sample. For some applications, it is not necessary to analyze all the proteins in the sample according to this invention since adequate information about the sample can be achieved with analyzing only a subset of the proteins in the sample. In alternate embodiments, all the proteins in the sample are analyzed. As described herein, the methods as described herein are equally amenable to detecting a fragment of the protein, rather than the entire protein. When only a portion of the protein is detected, information in the protein sequence databases can be effectively employed to guide detection according to the described methods.

[00125] Many proteins are proteolytically processed in cells and also contain specialized organelles for processing - such as the Golgi, Endoplasmic Reticulum, or Lysosomes. Other proteins may be proteolytically processed to be activated, such as Insulin. The ubiquitin/proteasome system has evolved to degrade proteins and many other proteases exist in cells to accomplish this function. Frequently; N-terminal Methionine amino acids are removed from some proteins, as well as signal sequences or other signatures of the proteins. Some protein degradation events may be necessary to accomplish the protein’s function, sub- cellular localization, transport or other biological mechanisms. The degraded forms or partially degraded forms in relation to the primary sequence likely exist in the sample and this property can be effectively employed during the choice of the appropriate condition to create any subsample. This invention is amenable to creating samples by sub-cellular fractionations of the source material, such as protein preparations from mitochondria, nucleus, membranes, or other.

[00126] In another aspect, the described methods include a protein fragmentation step, such as by treatment with a protease, or separating polypeptides comprising multi- subunit proteins in the sample. It is known in the art that treatment of proteins under extremes of pH, ion concentrations, temperature, chaotropic agents, ligands, etc., can disrupt secondary or tertiary structures, resulting in separation of polypeptides comprising the protein.

Fragmentation can be enhanced by supplying additional conditions to either the sample or any subsample of this invention. The fragmentation step is one example of the condition described in this invention and can be effectively combined with other approaches for subsample preparation described herein. [00127] Any condition applied to the sample or subsample of this invention can involve a range of values, including physical or chemical parameters. For instance, any value for pH, ionic concentration, temperature, chaotropic agent, ligands, chelators or other molecules. pH treatments can include a selected pH in the acidic or basic region, including neutral pH, or may involve a gradient in said regions. For the sake of example, and without limitation, gradients can be narrow, such as pH 1.0 to pH 1.5, or broad, such as, pH 1.0 to pH 4. Gradients can be continuous or discontinuous. Depending upon the selected condition to be employed, the range can be any that is desired, and can have any range size or position in the acidic to basic scale. In a preferred embodiment, the pH is in the 1.0 to 4.0 range. In alternate embodiments, the pH is in the 8.0 to 12.0 range. Likewise, ionic concentrations can vary over narrow or broad ranges, such as 50 to 150 mM, or 10 mM to 5M or greater, respectively. In preferred embodiments, the range of ionic concentrations is in the 50 mM to 250 mM range.

[00128] Any salts known in the art can be employed, including Sodium, Potassium, Magnesium, Iron, Cobalt, Nickel, or other mono-, di- or multi-valent metal salts. Ionic concentrations can also be varied with continuous or discontinuous gradients and fractions starting with sample or any subsample collected as multiple subsamples. In another aspect, proteins in sample or subsamples can be treated at varying temperatures or range of temperatures. For example, sample or subsample can be heated to 1 °C, 2 °C, 3 °C, or greater to collect proteins responsive to those temperatures as additional subsamples. In preferred embodiments, sample or subsample proteins can be treated at 37 °C, 42 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, 100 °C or greater. The selected temperature increments can be 0.1 °C or less, or greater than 5 °C, depending upon the specific needs of the experiment. Any specific temperature or range of temperatures can be applied to the proteins, continuously or discontinuously. According to this invention, it is not necessary to preserve the secondary, tertiary or quaternary structure of the protein or proteins in the sample or any subsample. In alternate embodiments, disrupting the structure can be the objective of the implementation. Likewise, sample or subsample proteins can also be treated at lower than ambient temperature, and such treatments can vary over narrow or wide ranges of selected temperatures.

[00129] Observational space:

[00130] As described herein, the methods include characterizing an observational space for observing proteins in the sample. The observational space can be a physical space, such as the region of a gel for fractionating proteins by size and/or charge; a device, such as a size fractionating column, including an HPLC column, an ion-exchange or hydrophobic matrix; a microfluidic device favoring the movement of proteins under the influence of an electric charge or other force; a column favoring the association of proteins due to their charge or hydrophobicity; a solid surface, including surfaces functionalized with an antibody, or molecules which interact with a protein in the sample, including a protein, a small or large molecule; or by separating in a mass spectrometer whereby proteins or the peptides therefrom are observed based on their mass-to-charge ratio, or a digital space for predicting the behavior of one or more proteins in a sample during any step of this invention.

[00131] In any aspect or embodiment, the observational window comprises separating proteins in the sample, more preferably a subsample, in a mass spectrometer. A number of mass spectrometric determinations of proteins have been described in the art, including Native MS, MS/MS, ESI, quadrupole, etc. Some of these implementations involve first separating proteins on a column, including reverse phase HPLC, subjecting proteins to an ionizing environment so as to fragment the protein, or by digesting the proteins with a protease. The described methods are amenable to employing any of these methods, alone or in combination.

[00132] Observation methods:

[00133] In certain embodiments, this invention describes repeatedly or sequentially observing proteins in a single (or substantially the same) observational space. For example, a first subsample prepared by treating sample proteins in a first buffer (comprising pH 7.0, 150 mM NaCl) and collecting the proteins associating with an ion exchange column or matrix is first observed. The proteins which do not associate with the matrix under these conditions, the eluate - the second subsample, can then be used to create a third subsample by treating under a second condition, such as a second buffer (under a different selected pH or salt concentration) and applied to the same ion exchange column or matrix. The new proteins which associate with the ion exchange column or matrix under the second condition comprise a different subsample, and can be detected. In this example, the observational space is the ion exchange column or matrix.

[00134] In accordance with methods described herein, altering the physical or chemical state of a protein or proteins using appropriate conditions affords the ability to observe proteins in a single observational space. For example, not all proteins in a sample or subsample possess the ability to associate with the example observation space above. For example, a protein possessing a net positive charge may be recovered in the first subsample using the above condition, however, the same protein which is phosphorylated or otherwise modified may not associate with the matrix, and be recovered in the second subsample.

Under appropriate conditions, such as, neutralizing the negative charge from the phosphate group by increasing the concentration of divalent metal ions, may restore the ability of the example phosphorylated protein to associate with the matrix. Likewise, a protein having an intrinsic net positive charge as well as the property of associating with a nucleic acid can be sequentially recovered in the observation space in tow steps - first, by treating sample under the first condition to recover protein not associated with the nucleic acid, and then treating the second subsample under a condition favoring release of the bound nucleic acid.

[00135] Other examples of fractionating sample proteins using different conditions but the same observation space include, protein isoforms, alternatively spliced forms, proteins associated with other proteins, proteins containing two or more polypeptides, proteins containing labile amino acids, protein complexes, proteins extracted from distinct cellular compartments, proteins associated with other molecules, including nucleic acids, small molecules, lipids, membranes, or cofactors. The described methods are amenable to forming complexes with sample proteins prior to applying a condition or separation means. The proteins participating in complex formation and the protein not participating in the complex formation can then be sequentially observed in two or more subsamples according to the methods of this invention. In general, any subsample is likely to yield a plurality of spots or data points corresponding to a plurality of proteins. Observed spots in any observation space constitute a signature.

[00136] In certain aspects or embodiments, the methods include comparing signatures across samples, such as, a cell type in two physiological states, a drug-treated and/or untreated cells, a native cell and a genome-edited cell, a native cell and the same cell type harboring a recombinant protein, and so on.

[00137] Alternately, proteins in the first test sample or subsample can be fractionated using a size fractionation column with a molecular weight cutoff property, to create two additional subsamples - the bound material and the eluate. In this example, the observational space is the size fractionation column. In a third example, the proteins in the first subsample can be fragmented prior to applying to the size fractionation column.

[00138] In any aspect or embodiment, two or more subsamples of this invention are observed in the same observational space, sequentially or in combination. For example, and without limitation, a first subsample is introduced in to the mass spec implementation of this invention and the pattern of peaks corresponding to the mass-charge (m/z) values of proteins in the subsample is acquired. The process can then be repeated with additional subsamples. A pattern of spots corresponding to proteins in the first subsample can also be generated by separating them on polyacrylamide gels, in one or two dimensions. Patterns of spots from different subsamples can be compared.

[00139] In any aspects or embodiments, the observational step comprises of observing a pattern - including, but not limited to, Mass Spectroscopy peptide signatures, spots on a 2- dimensional gel, contacting with an array, including antibody array, etc., generated by data relating to proteins in the subsample, hereinafter termed spots. The number of spots in the observational space is not important, but it is preferred that each of the spots is substantially separable from other spots in the same observational space so as to clearly observe a parameter - such as, Rf or m/z values, or signatures for substantially many of or all the separated proteins in the subsample. The anticipated result from this step is a spectrogram showing a multitude of peaks, each representing the m/z signature for one protein or peptide in the subsample. As such, in certain embodiments, the methods include applying the above or other methods known in the art commonly employed in mass spectroscopy or gel electrophoresis.

[00140] In any aspects or embodiments, a plurality of subsamples is sequentially analyzed in the observational space. In certain embodiments, each subsample is

characterized by a property used to prepare the subsample, such as size, charge,

hydrophobicity, etc. In a non-limiting manner, at least one property is different between different subsamples of this invention. For instance, a protein of size 7 kDa may be observed in the pattern from a first subsample comprising an eluate after treating a cell extract with a 10k size fractionation column under non-denaturing conditions. In a subsequent

implementation, the supernatant from the above implementation can be treated under a second condition, such as, denaturing under non-oxidizing conditions, and may lead to an observable spot of a 7 kDa protein or polypeptide in the mass spec implementation. In a further step, a third protein of substantially the same size or m/z value may be observable upon treating the supernatant from the second condition under oxidizing-denaturing conditions. Without limitation, the patterns of observed spots are likely distinct proteins or polypeptides, as characterized by the conditions of the experiment.

[00141] In any aspect or embodiment, a multitude of spots corresponding to proteins is observed in the observational space. The total number of spots appearing in the observational space is not critical, it can be any that allows the best combination of determining signatures or patterns relating to the spacing of the spots, ability to associate a value or attribute to a substantial number of the spots, one that provides an image of the pattern of spots.

[00142] Without limitation, the resulting pattern of spots from each subsample individually and collectively characterizes the subsample at each implementation, including experimental variables, such as the manner in which the subsample was created based on size, charge, hydrophobicity, or other feature of proteins. Without limitation, the manner of creation of subsample can be tuned up or down depending on the dynamic range of the observational method. The observational step can be varied by iterating subsamples, pooling subsamples or other approaches so as to allow the required or manageable number of proteins detected in the observational space.

[00143] In any aspect or embodiment, the observational space is one that maximizes the number of spots observable in the observational space, without excessive overlapping of the spots, while offering the ability of associating a value to substantially all the spots, such as a m/z value or Rf (log molecular weight versus relative mobility) in gel electrophoresis. The number of spots in the observational space can be 10 or fewer, 100 or fewer, 1000 or fewer, 10,000 or fewer or greater than 10,000. Multiplexed approaches involving mass spec are known in the art and routinely observe thousands of proteins. For example, it is not necessary to accurately determine every one of the spots appearing in the observational space, since sometimes observation of only a subset of the spots may be adequate to characterize the subsample, particularly when the experiment is focused on only a subset of the proteins, such as phosphorylated proteins or cysteine-disulfide linked polypeptides or one partner of a -S-S- linked protein. In this manner, the methods of this invention offer a high level of

customization of the methods of this invention, so as to meet the objectives of the

experiment.

[00144] Profiling:

[00145] In any aspect or embodiment described herein, the methods include a step of detecting and/or comparing the pattern of spots obtained with a subsample across two or more samples. This is termed the profiling approach. The samples may be closely related, such as, samples under two different biological, environmental, or other conditions, or from two different subjects, or a subject providing samples at two different times, or conditions. Other comparisons may include samples obtained from two or more distinct sources, such as human and mouse, two genetically distinct mice, or across species. Samples useful for comparisons also include normal versus disease state (or states), native versus recombinant, distinct disease states or stages, serum versus plasma; any two samples can be compared. The comparisons may also include the same sample under different storage conditions or time during which the sample was stored.

[00146] In the methods described herein, it is not necessary in the observational step to observe even a single difference in the pattern of spots when comparing two or more samples or subsamples, but is equally amenable to observing one or more differences.

[00147] Protein or peptide signatures:

[00148] In any aspect or embodiment, the methods as described herein observe protein signatures in a sample. In one embodiment, sample proteins are divided into two or more subsamples and the proteins in any subsample are observed in the observational space of this invention. The pattern of spots observable in the sample constitute a protein signature. In alternate embodiments, the pattern of spots in each of the subsamples constitutes a protein signature. A pattern of spots refers to any of several ways to observe proteins. For example, the subsample proteins can be fractionated on a gel by electrophoresis, including 2- dimensional gel, and stained with a suitable dye before imaging. The same can also be observed after transferring the proteins from the gels to a membrane and detecting by Western blotting, with one or more antibodies. In other embodiments, an ELISA approach can be used to detect proteins with antibodies, or Strep tavidin-HRP after tagging subsample proteins with a detectable label, such as biotin. A pattern of spots according to this invention also relates to protein sequence information from Mass Spectroscopic analysis of subsample proteins, with or without treating subsample proteins with a protease, such as Trypsin, prior to Mass Spec analysis. The methods as described herein are amenable to analyzing single proteins or a subset of the subsample proteins by excising bands or spots from gels and detecting with Mass Spectroscopy, ELISA, or other methods known in the art. It is not necessary to observe all the spots in the subsample using any of the above methods, it can be any that affords adequate information about the subsample as determined by the implementer of this invention. In yet another alternate embodiment, a single spot or a plurality of spots equaling less than the total number of observed spots in a sample or subsample constitutes a protein signature.

[00149] Detection:

[00150] In any aspect or embodiment, the methods described herein can use a variety of detection methods including, but not limited to, gel electrophoresis, column chromatography, antibody detection methods, affixing a label to sample proteins and detecting the label, and Mass Spectroscopy, which would be well known by the skilled artisan. The methods can also include appropriate imaging methods favoring detection of proteins.

[00151] Migration:

[00152] Migration includes physical movement of a protein or proteins, such as, under the influence of an electric current or other force, the preferential association or attachment of proteins to a label or surface, or other processes known in the art. Migration can also relate to the separation of two or more polypeptides comprising a sample protein, of a protein and an adduct, including a nucleic acid, small molecule, a cofactor, a binding protein, an antibody, or other moiety, or any that localizes a sample protein in the observation space. In some embodiments, migration also refers to a digital space comprising any selected parameter of the observation space.

[00153] The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

[00154] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. [00155] REFERENCES:

[00156] Patterson, S.D. 1998. ETsing MS fragment-ion data to identify proteins from large sequence databases, p. 127-135.

[00157] In L.M. Savage (Ed.), Proteomics, Integrating Protein-based Tools and Applications for Drug Discovery. International Business Communications, Southborough, MA.

[00158] Spahr, C.S., S.A. Susin, E.J. Bures, J.H. Robinson, M.T. Davis, M.D. McGinley, G. Kroemer, and S.D. Patterson. 2000. Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.

Electrophoresis 21:1635-1650.

Claims

Claims

1. A method of characterizing a cell or tissue type of interest, the method comprising the steps of:

(a) preparing a solid support that comprises a bait protein mixture from a first cell or tissue lysate from a subject, wherein the bait proteins are immobilized on a surface of the solid support;

(b) preparing at least one test cell or tissue lysate sample (at least one test sample), wherein the at least one test sample comprises a mixture of two or more prey proteins (prey protein mixture);

(c) contacting the solid support with the at least one test sample thereby allowing proteins from the bait protein and prey protein mixtures to form protein complexes on the solid support;

(d) eluting the bound test sample proteins from the complexes on the solid support;

(e) detecting the identity of the eluted proteins from the at least one test sample, thereby obtaining a protein signature for the at least one test sample; and

(f) characterizing the test cell or tissue type on the basis of the protein signature.

2. The method of claim 1, wherein the proteins in the bait protein mixture, the at least one test sample or both are at least one of native, partially denatured, fragmented, renatured, mutated, recombinantly or non-recombinantly expressed.

3. The method of any of claims 1 or 2, wherein the proteins in the bait protein mixture, the at least one test sample or both are not exogenously expressed or recombinant proteins.

4. The method of any of claims 1-3, wherein the proteins in the bait protein mixture, the at least test sample or both comprise whole cell protein extract or a fraction thereof.

5. The method of any of claims 1-4, wherein the at least one test sample comprises from 100 prey proteins to 25000 prey proteins or more.

6. The method of any of claims 1-5, wherein the proteins in the bait protein mixture and at least one test sample form a protein complex that comprises three or more proteins.

7. The method of any of claims 1-6, wherein each bait protein in the bait protein mixture is individually affixed to the solid support.

8. The method of any of claims 1-7, wherein the at least one test sample is from at least one of the same subject, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof.

9. The method of any of claims 1-8, wherein the method further includes repeating steps (b)-(e) with an additional test sample.

10. The method of claim 9, wherein the additional test sample is from at least one of the same subject, a different subject, the same cell or tissue type, a different cell or tissue type, a treated cell or tissue sample, or a combination thereof, as the at least one test sample from step (c).

11. The method of claim 9, wherein the additional test sample is from the same cell or tissue type as the at least one test sample from step (c) following treatment with an external stimuli.

12. The method of claim 11, wherein the external stimuli is drug treatment, temperature change, irradiation, physical agitation, contact with another cell or tissue, pH change, change in ion concentration, genetic modification of cell, an infected cell or tissue or any treatment which alters proteins or the biology of the cell.

13. The method of claim 1, wherein the protein signature is a protein interaction signature.

14. The method of claim 13, further comprising the step of profiling the protein signature with known protein interactors on a computer display.

15. The method of claim 1, wherein prior to step (d) the method includes a step of washing the solid support with buffer solution to remove non-specific binding proteins.

16. The method of claim 15, wherein step (d) includes eluting both the complexed proteins from the at least one test sample as well as the proteins bound to the solid support.

17. The method of claim 1, wherein step (a), step (b) or both includes treating the bait protein mixture, the at least one test sample or both to select or isolate one or more proteins of interest or providing a protein of interest.

18. The method of claim 17, wherein the treatment includes at least one of treatment with a chemical agent, treatment with an affinity reagent, modifying the pH, modifying the ionic concentration, adding or removing divalent cations, modifying the temperature, adding a reducing agent, adding a chaotropic agent, adding a denaturant, adding an additive or an enzyme or a combination thereof.

19. The method of any of claims 1-18, wherein the bait protein mixture from the first cell or tissue lysate is coupled directly to the surface of the solid support or is immobilized by binding to an affinity matrix on the surface of the solid support.

20. The method of any of claims 1-19, wherein the step of eluting comprises eluting selectively a population of the bound proteins from the complexes on the solid support.

21. The method of any of claims 1-20, wherein the bait protein mixture, prey protein mixture or both is from a cancer cell or cancer tissue.

22. The method of any of claims 1-21, wherein the proteins in the bait protein mixture are coupled to the solid support via cross-linking, hydrophobic interactions, an affinity matrix, or combination thereof.

23. The method of any of claims 1-22, wherein the step of contacting is performed under at least one of the following conditions:

a. low ionic strength,

b. medium ionic strength,

c. high ionic strength, d. pH < 5,

e. pH 5-8,

f. pH > 8

g. in the presence of divalent cations, Mg²⁺, Ca²⁺, Mn²⁺

h. at a temperature < 20 C,

i. at a temperature of 20-22 C,

j. at a temperature of > 22C, or

k. a combination thereof.

24. The method of any of claims 1-23, wherein the step of eluting selectively comprises eluting proteins using at least one of heat, ionic strength, pH, a chaotropic agent, small molecule inhibitor, allosteric binding agent, competitive binding agent, or a combination thereof.

25. The method of any of claims 1-24, wherein the detecting step comprises performing at least one of

trypsin digestion or fragmentation of proteins in the eluate, and performing m/z mass spectrometry identification of resulting protein or peptide fragments;

amino acid sequencing of protein or peptide fragments,

database searching,

gel electrophoresis,

detecting with an antibody or

a combination thereof.

26. The method of any of claims 1-25, wherein the method further comprises a step of identifying a parent protein based on the sequence of the peptide fragments, gel mobility, or antibody specificity or a combination thereof.

27. The method of claims 13 or 26, comprising the step of identifying at least one parent protein in the ubiquitin protein degradation pathway.

28. The method of claim 27, further comprising the step of characterizing proteins in the at least one test sample based on the signature of ubiquitin-protein degradation pathway proteins specific to the at least one test sample.

29. The method of claim 28, wherein the at least one test sample is characterized on the basis of the presence of from 2 to 1000 distinct proteins, including at least one ubiquitin- protein degradation pathway protein.

30. The method of any of claims 1-29, wherein subsequent to characterizing the at least one test sample, administering a therapeutic agent to the subject.

31. The method of any of claims 1-30, wherein the solid support is a resin, a membrane, a chip, a cartridge, a size fractionating column, an ion exchange column or matrix, or an affinity matrix.

32. The method of any of claims 1-31, further comprising the steps of

preparing an additional test sample,

producing an additional protein interaction signature according to claim 1 for the ubiquitin- protein degradation pathway proteins in the additional test sample, and

comparing the signatures to detect for a change, wherein a change is indicative of a modification of an altered state of the cells.

33. The method of any of claims 1-32, wherein the first cell or tissue lysate, the at least one test sample, or the additional test sample are from the same or different cell or tissue types, or the same or a different subject or a combination thereof.

34. The method of any of claims 1-33, wherein the method comprises changing or modifying at least one of a therapeutic agent, dosage of a therapeutic agent, a dosage regime or a combination thereof of a subject based on the protein signature.

35. The method of any of claims 1-34, wherein the proteins are derived from a cell, tissue, organ or components thereof.

36. The method of any of claims 1-35, wherein the at least one test sample is derived from a human, a bacterium, microbe, virus, plant or an animal.

37. The method of any of claims 27-29, wherein the ubiquitin-protein degradation pathway parent proteins comprise at least one of an E3 ligase, an E2 Ligase, a polyubiquitin, a USP, a cullin, a SUMO ligase, a deubiquitinase, a ubiquitin binding protein, a SCF protein, a proteasome protein, a peptidase, an F-box protein, or a combination thereof.

38. The method of claim 1 wherein the at least one test sample cell is derived from a B cell from a human source.

39. The method of claim 37, wherein the ubiquitin-protein degradation pathway parent proteins comprise at least one of AMFR, ASB18, ASB6, BRCC3, COP1, CUL4A, CUL4B, DCAF8, DTX3L, DTX4. HECTD1, HERC1, HERC2, JOSD1, LTN1, MARCH8, MINDY4, MYCBP2, PHC3, PPP1R11, RanBP2, RBBP6, RFFL, RNF111, RNF138, RNF168, RNF180, RNF181, RNF187, RNF213, RNF220, RNF25, RNF31, RNF40, RNF41, RNF6, RNF8, RPS27A, SIAH1, SIAH2, STUB1, TOPORS, TRAF2, TRAF6, TRIM32, TRIM37, TRIM41, TRIM24, TRIM72, TTC3, UBA2, UBA6, UBE2V2, UBLCP1, UBR4, UBR5, UBR7, UBXN6, UHRF2, UIMC1, UPL5, USP19, USP20, USP25, USP31, USP32, USP4, USP40, USP8, USP9X, MDM2, VHL, cereblon, XIAP or a combination thereof.

40. The method of claim 39, wherein the at least one test sample is characterized on the basis of the presence of at least two ubiquitin-protein degradation pathway proteins.

41. The method of claim 39, wherein the at least one test sample is characterized on the basis of the presence of from 3 to 10 ubiquitin-protein degradation pathway proteins.

42. The method of claim 39, wherein the at least one test sample is characterized on the basis of the presence of from 11 to 20 ubiquitin-protein degradation pathway proteins.

43. The method of claim 39, wherein the at least one test sample is characterized on the basis of the presence of from 21 to 50 ubiquitin-protein degradation pathway proteins.

44. The method of claim 1, wherein the protein interaction signature is indicative of protein interactions in vivo or in vitro.

45. The method of claim 25, wherein the amino acid sequence is indicative of the parent protein amino acid sequence.

46. The method of claim 45, wherein amino acid sequence is shorter than the full-length sequence or comprises a domain in a ubiquitin-protein degradation pathway protein.

47. The method of claim 46, wherein the domain comprises the interaction site of the ubiquitin-protein degradation pathway protein or that of an interacting protein.

48. The method of claim 1, wherein first cell or tissue lysate and the at least one test sample are from different physiological states of the same cell type.

49. The method of any of claims 1-48, wherein the cell or tissue lysate is obtained from a member selected from the group consisting of: a drug-treated cell, a gene edited cell, a diseased cell, a drug-resistant cell, a differentiating cell, a proliferating cell, and a

combination thereof.

50. The method of claim 37, wherein the ubiquitin-protein degradation pathway parent proteins comprise at least one of USP5, AMFR, OTUB2, OTUB 1, CALR, CAND1,

RAD23B, RPN1, UBA1, UBA52, UBE2R2, UBE2V1, USP14, USP15 or a combination thereof.

51. The method of claim 44 wherein the protein signature comprises at least one ubiquitin-protein degradation pathway parent protein from claim 50.

52. The method of claims 50 or 51 wherein the pathway further comprises at least one two proteins from the group consisting of: USP5, AMFR, OTUB2, OTUB 1, CALR, CAND1, RAD23B, RPN1, UBA1, UBA52, UBE2R2, UBE2V1, USP14, USP15, an ACTIN

PROTEIN, an ACTIN RELATED PROTEIN, AFDN, ANNEXIN PROTEIN, API5, ARF3, ARF4, ARF5, ARF6, ARHGEF2, BAG3, BCAP31, BRD4, CALM1, CANX, CASP14, CD36, CDC42, CFL1, CLIC1, CLTC, COPA, COPB 1, CPSF6, CSNK1E, CTNNB 1, CTNND1, DAD1, DDOST, DDX17, DDX39A, DDX39B, DDX3X, DDX5, DDX6, DERFl, DHX9, DIRAS2, DRG1, DSTN, DYNC1H1, DYNFFl, DYNFTl, ECPAS, EDF1, EPRS, ERH, ERLIN2, ERP44, EWSR1, EZR, FASN, FBL, FIGNL1, FN1, FYN, , G3BP1, GAN, GAR1, GCN1, GDI1, GSN, GTPBP1, A HISTONE PROTEIN, A HMG PROTEIN, A HNRNP PROTEIN, HRAS, A HSP PROTEIN, ILF2, ILF3, IP07, KIF5B, KIFC3, LSM3, M6PR, MAPK1, MAPRE3, MCCC2, MLF2, MYH14, MY09A, MY09B, NACA, NCL, NDRG1, NDUFB9, NONO, NOP56, NOP58, NUDT21, NUP210, OSTC, PABPC1, PABPC1L2A, PABPC4, PARK7, PCBP1, PDCD6, PDCD6IP, PDIA3, PDIA4, PDIA6, PFDN2, PFN1, PHB, PHB2, PICALM, PIN1, PKP3, PLEC, PLS3, POF1B, PPIA, PPIB, PPME1, PPP1CB, PPP2CA, PPP2CB, PPP2R1A, PROTEASOME PROTEIN, PTBP1, PTK7, PUR, A RAB PROTEIN, RAC1, RACK1, RALA, RALB, RAN, RANGAP1,

RAP2B, RBBP4, RBM14, RBMX, A RIBOSOMAL PROTEIN, RIT2, RNF39, S 100A14, S 100A7, SEC23A, SEC23B, SEC24C, SEC24D, SEC31A, SET, SF3B 1, SF3B2, SF3B3, SFN, SFPQ, SH3GL1, SHMT2, SLC1A5, SLC25A11, SLC25A3, SLC25A5, SLC25A6, SMARCA4, SND1, AN SNRNP PROTEIN, SNU13, SPATA5, SPTAN1, SPTLC1, SRI, SRSF1, SRSF2, SRSF3, SRSF6, SSR3, SSR4, STIP1, STT3A, STX12, SULT2B 1, SUM04, TAGLN2, TALDOl, TCP1, TECR, TF, TFG, TIAL1, TMCOl, TMED10, TMED9, TOPI, TOP2B, TPD52L2, TPT1, TSPO, A TUBULIN PROTEIN, TUFM, TWF1, VAMP2, VAPB, VCP, VDAC1, VDAC2, VIM, VPS26A, VPS29, XPOl, XP07, XPOT, XRCC6, YBX3, YOD1, and combinations thereof.

53. The method of claims 34 or 52 wherein the cells wherein the therapeutic agent is selected from the group consisting of: JQ1, AZD5153, and I-BET151.