Diagnostic method based on large scale identification of post-translational modification of proteins

Abstract

Methods for the large scale identification of post-translational modification states of proteins and enzyme activities for carrying out post-translational modification reactions involve the analysis of functional extracts from fresh and frozen samples using protein arrays. The methods and kits of the present invention can be used to analyze and characterize compounds for their effects on post-translational modifications and their pathways. The methods and kits can also be used to diagnose and characterize a wide variety of diseases and medical conditions, including cancer, neurodegenerative diseases, immune diseases, infectious diseases, genetic diseases, metabolic conditions, and drug effects using cells or body fluids of a patient.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application under 35 U.S.C. § 120 of U.S. Ser. No. 12 / 696,866 filed on Jan. 29, 2010, which is a continuation-in-part application under 35 U.S.C. § 120 of an International Application PCT / US09 / 005670, filed Oct. 19, 2009, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 61 / 196,461, filed Oct. 17, 2008, the contents of which are hereby incorporated by reference in their entireties.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant No. GM039023 awarded by The National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted via EFS-WebPatent Center and is hereby incorporated by reference in its entirety. Said ASCIIXML copy, created on Feb. 4, 2010Sep. 26, 2023, is named 28060668.txt002806-066834USRE_SL.xml, and is 1,2222,876 bytes in size.

[0004] This is an application for reissue of U.S. Pat. No. 11,333,668. BACKGROUND OF THE INVENTION

[0005] Post-translational modification (PTM) of proteins has been studied largely using purified systems or whole cells. The analysis of protein PTM in cell extracts as well as extracellular fluids is both theoretically and empirically problematic. For example, both ubiquitination and phosphorylation, common examples of PTM, are very rapidly reversed, and this reversal requires no energy input or special conditions, aside from the actions of isopeptidases and phosphatases. Moreover, classical biochemical methods such as Western blot do not work well for concentrated mixtures of proteins, because the modified protein bands spread throughout the electrophoretic gel, and in complex samples, such as a cell extract or a blood plasma sample, many protein species would overlap, making protein identification difficult or impossible. Specifically, genome-wide methods for detecting PTM alterations are still in their infancy and largely depend on the interactions of biochemically purified systems. Chemical methods such as mass spectrometry cannot distinguish ubiquitin and polyubiquitin chains, yet only the latter are critical for protein degradation. A further limitation of such classical biochemical methods is that cryopreserved specimens which can be more readily available or are more logistically easy to procure cannot be used for most of these analyses and may have altered representation of the physiological condition. Furthermore, MS methods do not analyze the activity / function of a specific tissue / sample and its content but rather identifies the abundance of certain proteins in it. Thus, the complexity of the tissue and the dynamic range of different protein level are often limiting their detection.

[0006] In recent years, our understanding of posttranslational modifications and their implication for human diseases have greatly increased. In Alzheimer's disease (25) and Parkinson's disease (26-28) the ubiquitination of proteins has been shown to play a pivotal role in the regulation of cellular processes and human pathologies. Although the role that ubiquitination plays in tumorigenesis is still poorly understood, cases of ubiquitin ligases showing relationships with oncogenesis were recently described (29-31). Thus, systematic assays for the screening, including diagnostic screening, of ubiquitinated or other post-translationally modified proteins remain limited.BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides methods and kits for the systematic and large scale determination of protein PTMs and the enzyme activities that catalyze them. The methods entail incubating protein microarrays or another protein array format with cell extracts or fluids from a subject, performing specific PTM reactions on the microarrays, and detecting protein modification states of specific proteins. The methods according to the invention overcome obstacles associated with classical biochemical techniques by performing PTM reactions on protein microarrays with biological samples, such as patient materials, whose physiological state is preserved, appropriately supplemented, if so desired, with limiting PTM reaction components, and make it possible for the first time to rapidly screen patient samples for activities that modulate PTM states related to disease, and to rapidly screen for test agents that modulate PTM or PTM alteration pathways.

[0008] Accordingly, in one aspect, described herein is a method of identifying at least one post-translational modification (PTM) or PTM alteration on at least one protein, the method comprising the steps of:

[0009] (a) contacting a functional cell extract with a solid state array, the array comprising an ordered plurality of proteins under conditions that allow PTM to occur or that allow PTM to be modified;

[0010] (b) establishing at least one PTM reaction or PTM alteration reaction thereof on the array, whereby the reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and

[0011] (c) detecting the at least one PTM or PTM alteration by detecting a signal from the array thereby identifying the PTM or PTM alteration on the at least one protein.

[0012] In one embodiment of this aspect, the method further comprises identifying the effect of a test agent on the PTM or PTM alteration comprising the additional steps of:

[0013] (a) contacting the functional cell extract with a test agent;

[0014] (b) establishing at least one PTM reaction or PTM alteration on the array in the presence of the test agent, whereby the PTM reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and

[0015] (c) detecting the at least one PTM or PTM alteration and comparing the PTM reaction or PTM alteration reaction with a parallel reaction where a control agent has been added thereby allowing for detection of an effect of the test agent on at least one PTM or PTM alteration.

[0016] In one embodiment of this aspect, an increase in the signal from the array compared to a background or the reaction with a control is indicative of increased PTM. In another embodiment of this aspect, a decrease in the signal from the array compared to a background or the reaction with a control is indicative of PTM alteration.

[0017] In one embodiment of this aspect, the detecting is performed using an antibody or antigen-binding fragment thereof, a natural or recombinant ligand, a small molecule, a modifying moiety, or a biochemical analysis capable of detecting the PTM or PTM alteration. In some embodiments, the antibody or antigen-binding fragment thereof, the natural or recombinant ligand, the small molecule, or the modifying moiety is labeled with a tag. In some such embodiments, the tag is a fluorescent molecule, a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a chemiluminescent moiety, magnetic particle, bioluminescent moiety, or peptide. In some embodiments, the biochemical analysis is performed using mass spectroscopy, peptide mapping, or amino acid sequencing.

[0018] In one embodiment of this aspect, the functional cell extract is not diluted prior to the contacting with the solid state array. In one embodiment of this aspect, the functional cell extract is concentrated prior to the contacting with the solid state array.

[0019] In another embodiment of this aspect, the functional cell extract is obtained from a frozen or cryopreserved sample.

[0020] In another embodiment of this aspect, an additional cellular energy source in the form of ATP is provided to the functional cell extract.

[0021] In another embodiment of this aspect, the array comprising a plurality of proteins, comprises at least one protein, protein fragment or peptide attached to the array without an added tag.

[0022] In another embodiment of this aspect, the array comprising a plurality of proteins comprises at least one protein, protein fragment or peptide attached to the array with a C-terminal or N-terminal tag.

[0023] In another embodiment of this aspect, the functional cell extract is derived from a specified cellular compartment. In one embodiment, the cellular compartment is nucleus. In one embodiment, the cellular compartment is cytosol. In one embodiment, the cellular compartment is mitochondria.

[0024] In another embodiment of this aspect, the functional cell extract is derived from a biological sample. In one embodiment, the biological sample is selected from the group consisting of saliva, whole blood, serum, plasma, urine, cerebrospinal fluid, peritoneal fluid, chorionic villus, placenta, solid tissue, amniotic fluid, a cell sample, and a tissue culture sample.

[0025] In one embodiment of this aspect, the PTM is selected from the group consisting of ubiquitination, phosphorylation, glycosylation, sumoylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, neddylation, OClcNAc, ADP-ribosylation, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, carboxylation, and any combination thereof.

[0026] In one embodiment of this aspect, the PTM alteration is selected from the group consisting of deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufmylation, and any combination thereof.

[0027] In another embodiment of this aspect, the solid state array is selected from the group consisting of protein arrays on microchips, ELISA plates with immobilized proteins attached on the plates, protein-coated beads, and microfluidic chips coated with desired proteins.

[0028] In another embodiment of this aspect, 2-10 PTM or PTM alterations thereof are identified simultaneously.

[0029] In one embodiment of this aspect, and all such aspects described herein, the invention utilizes protein microarrays or other array formats of proteins together with appropriately supplemented functional cell extracts or body fluid samples to study the role of PTM in the presence and progression of many types of disease and many aspects of cellular function. Certain PTM states are mechanistically involved in cellular protein turnover, and consequently PTM states can be correlated with diseases related to protein turnover, such as, for example, Alzheimer's disease and other neurodegenerative diseases, and diseases related to regulation of the cell cycle, such as cancer.

[0030] In one aspect, the invention provides a method of identifying an altered PTM state of a protein in a patient. The method includes contacting a functional extract of a sample from the patient with a microarray containing an ordered plurality of proteins that represent proteins in the patient, establishing conditions for a specific PTM reaction in the extract, and determining the level of PTM of one or more proteins in the microarray. The presence or absence, or the observed level, of PTM of proteins in the microarray is then compared with the level of PTM of the corresponding proteins in a control sample, so that altered PTM states of proteins are identified that are expected to be similarly altered in the patient.

[0031] Another aspect of the invention is a method of identifying a protein PTM enzyme activity in a patient. The method includes contacting a functional extract of a sample from the patient with an array comprising an ordered plurality of proteins that represent proteins in the patient, and identifying post-translationally modified proteins in the array. The presence or absence, or the relative amount, of a PTM enzyme activity in the patient can be inferred from the protein posttranslational modifications observed in the array. The presence or absence, or the relative amount, of a corresponding PTM state produced by the enzyme activity in the patient may also be inferred from the results obtained with this method.

[0032] Still another aspect of the invention is a method of diagnosing a disease or medical condition in a patient. The method includes contacting a functional extract of a sample from the patient with a microarray containing an ordered plurality of proteins that represent proteins in the patient and identifying post-translationally modified proteins in the microarray to obtain a PTM state data set. The data set can serve as a signature or profile of protein PTMs in the patient as well as of the enzymes producing them. The data set is then compared with a standard data set that includes PTM state data diagnostic for the disease or medical condition and, based on the comparison, the disease or medical condition is diagnosed in the patient.

[0033] Yet another aspect of the invention is a method of identifying a set of biomarkers for a disease or medical condition. The method includes comparing the PTM profile of one or more patients having the disease or medical condition with similar profiles from one or more control subjects who do not have the disease or medical condition. The profiles are obtained by separately contacting functional extracts from the patients and control subjects with an array containing an ordered plurality of proteins, such as proteins encoded by the human genome, and determining the level of PTM of one or more proteins in the array. The presence or absence, or the observed level, of PTM of proteins in the array for the patients is then compared with the presence or absence or level of PTM of the corresponding proteins for the control subjects. A set of biomarkers is formed from proteins of the patients whose level of PTM is altered compared to control levels.

[0034] In a further aspect, the invention provides a kit for the diagnosis of a disease or medical condition, or the characterization of the effects of a drug, by the analysis of a PTM state of a protein in a patient sample. The kit includes a standard containing one or more functional extracts capable of producing a known pattern of protein PTM states on a protein microarray or in another array format. The kit also is adapted for, and contains instructions for, carrying out one of the above described methods. Optionally, the kit further contains a protein microarray, or a reagent such as a substrate, an enzyme, an enzyme inhibitor, a drug, or one or more antibodies. When applied with a method according to the invention, the standard produces a pattern of protein PTM that is diagnostic for a disease or medical condition, or the effects of a drug.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings.

[0036] FIG. 1A presents a schematic illustration of a PTM reaction carried out on a protein microarray using a functional extract from a patient sample. FIG. 1B shows a schematic illustration of the use of a PTM reaction on a protein microarray to diagnose a disease in a patient sample. The inset shows a reaction scheme common to ubiquitin-like modifiers, and the inset at the right shows example E1 and E2 enzymes for several ubiquitin-like modifiers.

[0037] FIG. 2A shows the degradation of 35S-labeled securin, added as a control to functional extracts, as a function of time at selected points during the cell cycle. The reactions were stopped at the indicted times by the addition of sample buffer and were then analyzed by SDS-PAGE and autoradiography. The star (*) labeled lanes reflect the state of the extracts at the time when incubation on the protein microarrays were stopped. FIG. 2B is a schematic illustration of the use of a protein microarray for the detection of posttranslational modifications. An example of one block / subarray out of the 48 on each chip is given (16 rows×16 columns). FIG. 2C is a schematic description of the steps of using a protein microarray for the detection of PTMs and PTM alterations.

[0038] FIG. 3A shows the distribution of signal intensity minus background values of all the spots on a protein microarray after detection of polyubiquitinated proteins. Reactivities were divided into 100 equally-sized bins, and the number of spots (y-axis) at different intensity levels (x-axis) of CP-released (left) and APC-inhibited (right) cell extracts was plotted. The inset represents a 20× magnification of the positive signals where the y-axis ranges between 0 and 250 and the x-axis ranges between 0 and 45,000. In FIG. 3B the reactivity level of 13 known APC substrates (dots) was compared to the reactivity level of the ‘buffer’ spots located in the same subarray (stars). The reactivities were then compared using a two-sample t-test to determine their significance, and the p-values were labeled below each substrate. FIG. 3C shows scatter plots of the positive signal intensities on each chip. The plots show the variability between two biological replicates (black dots; x-axis: CP-released, y-axis: CP-released) vs. the variability between signals from two different conditions (red dots; x-axis: APC-inhibited, y-axis: CP-released).

[0039] FIG. 4A shows analysis by SDS-PAGE (4-15% gels) and autoradiography of 35S-labelled substrates (Nek9, Calm2, RPS6KA4 and cyclin G2) added to CP synchronized HeLa S3 extracts with and without the addition of the APC-inhibitor emi1. FIG. 4B shows a similar analysis in which 35S-labelled p27 was added to CP synchronized HeLa S3 extracts with the addition of UbcH10, DN-UbcH10, or MG-132, or Emi1; the bottom panel shows the change in stability of p27 under this condition. The top panel is the same gel exposed for 4 days (long exposure) to detect p27-conjugated ubiquitin chains.

[0040] FIGS. 5A and 5B show the results of experiments to test the recognition of polyubiquitinated proteins with FK1 antibody.

[0041] FIG. 6 shows the distribution of signal and background levels observed on four representative protein microarrays.

[0042] FIG. 7 shows the signal-to-noise ratio for all spots on a protein microarray chip.

[0043] FIG. 8 shows the signal-background values for the buffer spots on five representative protein microarrays.

[0044] FIG. 9 shows the levels of the indicated endogenous proteins in functional extracts as a function of time as detected by Western blotting.

[0045] FIGS. 10A and 10B show the signal intensity distribution of all the spots on a protein microarray. FIG. 10A shows the results for a CP-released extract, and FIG. 10B shows the results for an APC-inhibited extract.

[0046] FIG. 11 shows human proteins that were significantly ubiquitinated by enzymes present in cerebrospinal fluid (CSF) from a patient with brain tumor.

[0047] FIG. 12 shows a Western blot of normal human CSF proteins that were polyubiquitinated using enzyme activity in CSF.

[0048] FIG. 13 shows the results of ubiquitination of a microarray of human proteins using normal human CSF. The number of ubiquitinated proteins detected is represented as a function of the fold increase of fluorescence over background.

[0049] FIG. 14 shows human proteins detected on a microarray as polyubiquitinated by enzymes present in two normal human CSF samples. The proteins shown revealed a fluorescence signal at least 50-fold over background.

[0050] FIG. 15 shows the fluorescence signal obtained for differentially modified proteins on a microarray after the indicated PTM reactions using extracts of mitotic checkpoint arrested and released HeLa S3 cells.

[0051] FIG. 16 presents a Venn diagram illustrating the relationships among protein targets found to be modified by different ubiquitin-like modifiers.DETAILED DESCRIPTION OF THE INVENTION

[0052] The inventors have developed methods that permit the rapid and large-scale diagnostic screening of altered protein PTM and PTM alteration states and related enzyme activities correlated with disease. The methods involve, in part, applying concentrated cell extracts or biological fluid samples from a subject to protein microarrays and appropriately supplementing them to carry out one or more specific PTM or PTM alteration reactions. Specifically, one or more PTM or PTM alterations are then detected by labeling the modified proteins and scanning the array.

[0053] Patterns of post-translational changes in certain polypeptides are known to correlate with certain diseases, such as Alzheimer's disease and cancer (see, for example, Table 3). While the altered polypeptides themselves may be detectable in extracellular fluids or cell extracts, and could be useful in diagnosing disease and monitoring its progression, an easier alternative to looking for the modified proteins themselves is to assay for the activity of specialized enzymes that make the modifications and are present in such fluids or extracts. Such assays are the focus, in part, of this invention. Assaying for such activities requires, in addition to the enzyme itself or enzymes themselves, which is / are supplied by the biological sample, such as a patient sample, the presence of one relevant cofactors and appropriate substrates. A PTM or PTM alteration activity assay can, for example, be used not only to diagnose a disease state, it can also be used to identify candidate biomarkers of diseases in biological fluid samples and cell extracts prepared from patient samples, and to test the effects of test agents on PTM or PTM alteration pathways, for applications such as drug design and discovery. Knowledge of the modified target proteins in a disease provides intrinsically important information about the altered post-translational process that occurs in the disease and its role in the disease.

[0054] Covalently modified proteins, such as polyubiquitinated, ubiquitinated, phosphorylated, glycosylated, sumoylated, acetylated, S-nitrosylated or nitrosylated, citrullinated or deiminated, neddylated, OClcNAc-added, ADP-ribosylated, methylated, hydroxymethylated, fattenylated, ufmylated, prenylated, myristoylated, S-palmitoylated, tyrosine sulfated, formylated, and carboxylated proteins are hard to identify by the standard biochemical technique of gel electrophoresis, because the modified protein bands spread throughout the gel. Identifying the converse alteration of a PTM, such as, for example, deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, deS-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, and deamidation is similarly difficult to detect using such standard biochemical methods. In a complex sample like a functional cell extract or biological sample, such as an undiluted or concentrated body fluid, many protein molecular species would overlap, making identification of specific modified proteins difficult or impossible. The high concentration and large number of different proteins in patient samples such as cell or tissue extracts, and body fluids such as blood plasma or CSF, generally require additional processing steps to separate the sample into different fractions or to purify certain molecular components prior to analysis. In contrast, with the present methods described herein, a PTM or PTM alteration reaction is performed directly on a solid state array, such as a protein microarray, or any other array format wherein the location of each protein is known. The known physical location of the protein on the array, rather than its electrophoretic mobility in a gel, is used to identify the target. Combined with the use of antibodies that have binding specificity for particular PTM or PTM alteration states, such as polyubiquitinated vs. monoubiquitinated proteins, or combined with the use of any labeled modifying moiety, the use of protein arrays greatly simplifies the problem of identifying specific PTM or PTM alteration states on specific proteins, and the use of multiplex formats, such as microarrays, also makes possible the simultaneous analysis of thousands of proteins. Thus, the present invention overcomes previous obstacles to identifying altered PTM or PTM alteration states and altered activity of enzymes that produce PTM or PTM alteration in a patient and brings PTM and PTM alteration analysis into a realm where it is possible for the first time to diagnose disease in a clinical setting.

[0055] Accordingly, in one aspect, described herein is a method of identifying at least one post-translational modification (PTM) or PTM alteration on at least one protein, the method comprising the steps of:(a) contacting a functional cell extract with a solid state array, the array comprising an ordered plurality of proteins under conditions that allow PTM to occur or that allow PTM to be modified;(b) establishing at least one PTM reaction or PTM alteration reaction thereof on the array, whereby the reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and(c) detecting the at least one PTM or PTM alteration by detecting a signal from the array thereby identifying the PTM or PTM alteration on the at least one protein.

[0056] In one embodiment of this aspect, the method further comprises identifying the effect of a test agent on the PTM or PTM alteration comprising the additional steps of:(a) contacting the functional cell extract with a test agent;(b) establishing at least one PTM reaction or PTM alteration on the array in the presence of the test agent, whereby the PTM reaction results in at least one PTM or PTM alteration of at least one protein on the array through the activity of one or more enzymes present in the cell extract; and(c) detecting the at least one PTM or PTM alteration and comparing the PTM reaction or PTM alteration reaction with a parallel reaction where a control agent has been added thereby allowing for detection of an effect of the test agent on at least one PTM or PTM alteration.

[0057] As used herein, an “agent” for use in the methods described herein refers to any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In certain embodiments, agents are small molecules having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and / or property, or can be selected from a library of diverse compounds.

[0058] As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0059] In such embodiments, the effects of one or more test agents that modify specific PTM or PTM alteration pathways can be determined using the methods described herein. The ability to rapidly screen one or more test agents for effects on a multitude of specific PTM or PTM alteration reactions simultaneously is useful for drug design and discovery purposes. As defined herein, a test agent that modifies or modulates a specific PTM or PTM alteration pathway is one that causes a detectable change in a PTM or PTM alteration reaction mediated by a functional cell extract, such as, changing the kinetics of the reaction (increase or decrease) or preventing the reaction from occurring entirely. In some embodiments, the test agent can replace a missing component of the functional cell extract, such that a PTM or PTM alteration reaction occurs, which did not occur in the absence of the test agent. In such embodiments, the test agent acts to replace or modulate a component of the PTM or PTM alteration pathway. The ability to rapidly and simultaneously screen for the effects of a test agents on PTM or PTM alteration pathway is useful for high-throughput applications, such as screening of compounds for drug discovery applications.

[0060] In another embodiment, the methods described herein comprise detecting the PTM or PTM alteration using one or more agents capable of specifically detecting the PTM or PTM alteration. Agents specific for detecting the PTM or PTM alteration include, but are not limited to, antibodies or antigen-binding fragments thereof, natural or recombinant ligands, small molecules; nucleic acid sequence and nucleic acid analogues; intrabodies; aptamers; and other proteins or peptides; and a modifying moiety. In some embodiments, the detecting comprises the use of one or more antibodies which are directly labeled with a tag. In other embodiments, the detecting comprises the use of one or more antibodies than can be detected using a secondary antibody. In some embodiments, the secondary antibody is directly labeled with a tag. In other embodiments, the secondary antibody is detected using a tertiary antibody directly labeled with a tag. In other embodiments, one or more biochemical methods can be used for detecting PTM or PTM alterations. In such embodiments, the biochemical methods can include, but are not limited to, mass spectroscopy, peptide mapping, and amino acid sequencing.

[0061] In some embodiments of this aspect and all aspects described herein, the preferred agents specific for detecting the PTM or PTM alteration are antibody agents that specifically bind the PTM or PTM alteration, and can include polyclonal and monoclonal antibodies, and antigen-binding derivatives or fragments thereof. Well-known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab′)2 fragment. Methods for the construction of such antibody molecules are well known in the art. Accordingly, as used herein, the term “antibody” refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. “Antigen-binding fragments” include, inter alia, Fab, Fab′, F(ab′)2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc′, F(ab′) 2 and Fv are employed with standard immunological meanings [Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)]. Such antibodies or antigen-binding fragments specific for CD31, CD105, CD105, CD44, and Sca-1 are available commercially from vendors such as R&D Systems, BD Biosciences, e-Biosciences and Miltenyi, or can be raised against these modifications by methods known to those skilled in the art.

[0062] In some embodiments of the aspects described herein, an agent specific for a PTM or PTM alteration, such as an antibody or antigen-binding fragment thereof, a natural or recombinant ligand, a small molecule, or a modifying moiety, is directly labeled with a tag to facilitate the detection of the modification. The terms “label” or “tag”, as used herein, refer to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific modification in a biological sample. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, peptide tags (c-Myc, HA, VSV-G, HSV, FLAG, V5 or HIS) and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to identify the PTM or PTM alteration. In some embodiments of the aspects described herein, the modification moiety itself may be labeled directly. For example, one can use a radioactive label or a florescent label so that the protein modification can be read directly (or in combination with other modifications) without the use of antibodies. Naturally, also antibodies may be labeled to assist in their direct detection.

[0063] The terms “labeled antibody” or “tagged antibody”, as used herein, includes antibodies that are labeled by detectable means and include, but are not limited to, antibodies that are fluorescently, enzymatically, radioactively, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS, which can be detected using an antibody specific to the tag, for example, an anti-c-Myc antibody. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Non-limiting examples of fluorescent labels or tags for labeling the antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7.

[0064] In some embodiments of the methods described herein, a PTM comprises ubiquitination, phosphorylation, glycosylation, sumoylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, neddylation, deimination, OClcNAc, ADP-ribosylation, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, carboxylation, and any combination thereof. In some embodiments, a PTM consists essentially of ubiquitination, phosphorylation, glycosylation, sumoylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, neddylation, OClcNAc, ADP-ribosylation, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, carboxylation, and any combination thereof. In some embodiments, a PTM consists of ubiquitination, phosphorylation, glycosylation, sumoylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, neddylation, OClcNAc, ADP-ribosylation, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, carboxylation, and any combination thereof.

[0065] In some embodiments of the methods described herein, a PTM alteration comprises deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufinylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof. In some embodiments, a PTM alteration consists essentially of deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof. In some embodiments, a PTM alteration consists of deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, de-S-nitrosylation or denitrosylation, decitrullination or dedeimination, deneddylation, removal of OClcNAc, de-ADP-ribosylation, demethylation, de-hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof.

[0066] As used herein, the term “post-translational modification” or “PTM” refers to a reaction wherein a chemical moiety is covalently added to or non-covalently binds to protein. As used herein, the term “PTM alteration” refers to a reaction wherein a chemical moiety covalently attached to or non-covalently bound to a protein is removed or altered (maybe in chain topology, different PTM combinations, etc). “Covalent bonding,” as used herein, refers to the form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, and other covalent bonds. Covalent bonding includes many kinds of interactions, including, but not limited to, α-bonding, π-bonding, metal to non-metal bonding, agostic interactions, and three-center two-electron bonds. “Non-covalent bonding,” as used herein, refers to the type of chemical bond, typically between macromolecules, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. Noncovalent bonds are critical in maintaining the three-dimensional structure of large molecules, such as proteins and nucleic acids, and are involved in many biological processes in which large molecules bind specifically but transiently to one another. Examples of noncovalent interactions include, but are not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces, i.e. “London dispersion forces”, and Dipole-dipole bonds.

[0067] Many proteins can be post-translationally modified through the covalent addition or transient non-covalent binding of a chemical moiety (also referred to herein as a “modifying moiety”) after the initial synthesis (i.e., translation) of the polypeptide chain. Such chemical moieties usually are added by an enzyme to an amino acid side chain or to the carboxyl or amino terminal end of the polypeptide chain (i.e., PTM), and may be cleaved off by another enzyme (i.e., PTM alteration). Single or multiple chemical moieties, either the same or different chemical moieties, can be added to or bound to a single protein molecule. PTM of a protein can alter its biological function, such as its enzyme activity, its binding to or activation of other proteins, or its turnover, and is important in cell signaling events, development of an organism, and disease. Examples of PTM covered by the methods of the invention described herein include, but are not limited to, ubiquitination, phosphorylation, sumoylation, neddylation, ADP-ribosylation, glycosylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, the addition of OClcNAc, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, and carboxylation. In some embodiments, a PTM can include both a covalent addition and non-covalent binding of a chemical moiety to a protein. For example, small ubiquitin-related modifiers (SUMOs) can be both covalently conjugated to a protein, and transiently non-covalently bound to the same protein to mediate different effects. In such embodiments, the covalent conjugation and non-covalent binding require different sequence motifs.

[0068] Similarly, a PTM alteration can involve removal of a covalently conjugated or a non-covalently bound chemical moiety. Examples of PTM alteration covered by the methods of the invention described herein include, but are not limited to, deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, deS-nitrosylation, denitrosylation, decitrullination or dedeimination, deneddylation, de-ADP-ribosylation, removal of OClcNAc, demethylation, de-hydroxylation, defattenylation, deufmylation, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, and deamidation.

[0069] As used herein, “ubiquitination” or “ubiquitylation” refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The ubiquitylation cascade is started by the E1 enzyme. The amino acid sequence of human ubiquitin is:

[0070] (SEQ ID NO: 1)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG.

[0071] As used herein, removal of one or more ubiquitin molecules is known as “deubiquitination.”

[0072] As used herein, “phosphorylation” refers to the addition of a phosphate (PO4) group to a protein or other organic molecule. As used herein, “dephosphorylation” refers to the removal of a phosphate group from a protein or other organic molecule.

[0073] As used herein, “sumoylation” refers to the process whereby Small Ubiquitin-like Modifier or “SUMO” proteins are covalently attached to other proteins in cells to modify their function. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. As defined herein, “desumoylation” refers to the process whereby SUMO proteins are removed from proteins in cells.

[0074] As used herein, “neddylation” refers to the process by which the ubiquitin-like protein Nedd8 is conjugated to its target proteins. This process is analogous to ubiquitination, although it relies on its own E1 and E2 enzymes. As used herein, “deneddylation” refers to the process by which the ubiquitin-like protein Nedd8 is unconjugated from its target proteins.

[0075] As used herein, “ADP-ribosylation” refers to the PTM of proteins that involves the addition of one or more ADP and ribose moieties. As used herein, “de-ADP-ribosylation” refers to the removal of one or more ADP and ribose moieties.

[0076] As defined herein, “glycosylation” refers to the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules. For the methods described herein, glycosylation includes N-linked glycosylation, O-linked glycosylation (O—N-acetylgalactosamine (O-GalNAc), O-fucose, O-glucose, O—N-acetylglucosamine (O-GlcNAc), O—N-acetylglucosamine, O-mannose, Collagen Glycosylation, Hydroxyproline Glycosylation, Glycosylation of Glycogenin, Glycosylation of Ceramide, Proteoglycans), phospho-Serine Glycosylation and C-mannosylation. As defined herein, “deglycosylation” refers to the enzymatic process that removes saccharides attached to proteins, lipids, or other organic molecules.

[0077] As used herein, “acetylation” (or in IUPAC nomenclature “ethanoylation”) refers to the reaction that introduces an acetyl functional group into a chemical compound, and includes N-alpha-terminal acetylation and lysine acetylation. As used herein, “deacetylation” (or in IUPAC nomenclature “de-ethanoylation”) refers to the reaction that removes an acetyl functional group from a chemical compound.

[0078] As defined herein, “S-nitrosylation” or “nitrosylation” refer to the addition of a nitroso group to a sulfur atom of an amino acid residue of a protein. As defined herein, “de-S-nitrosylation” or “de-nitrosylation” refer to the removal of a nitroso group from a sulfur atom of an amino acid residue of a protein.

[0079] As used herein, “citrullination” or “deimination” are the terms used for the post-translational modification of the amino acid arginine in a protein into the amino acid citrulline. As used herein, “decitrullination” or “de-deimination” are the terms used for the removal of the amino acid citrulline from a protein.

[0080] As used herein, “methylation” is the term used to denote the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with specifically a methyl group. Protein methylation typically takes place on arginine or lysine amino acid residues in the protein sequence. Arginine can be methylated once (monomethylated arginine) or twice, with either both methyl groups on one terminal nitrogen (asymmetric dimethylated arginine) or one on both nitrogens (symmetric dimethylated arginine) by peptidylarginine methyltransferases (PRMTs). Lysine can be methylated once, twice or three times by lysine methyltransferases. As used herein, “demethylation” refers to the removal of a methyl group from a protein.

[0081] As used herein, “hydroxylation” refers to the chemical process that introduces one or more hydroxyl groups (—OH) into a compound (or radical) thereby oxidizing it. The principal residue to be hydroxylated in proteins is proline. The hydroxylation occurs at the Cγ atom, forming hydroxyproline (Hyp). In some cases, proline may be hydroxylated instead on its Cβ atom. Lysine may also be hydroxylated on its Cδ atom, forming hydroxylysine (Hyl). As used herein, “dehydroxylation” refers to the chemical process that removes one or more hydroxyl groups (—OH) from a protein.

[0082] As used herein, “ufmylation” refers to the process whereby the ubiquitin-like modifier Ufm-1 is covalently attached to a protein. As used herein, “deufinylation” refers to the process whereby the ubiquitin-like modifier Ufm-1 is removed from a protein.

[0083] As used herein, “fattenylation” refers to the process whereby the ubiquitin-like modifier FAT10 is covalently attached to a protein. As used herein, “defattenylation” refers to the process whereby the ubiquitin-like modifier FAT10 is removed from a protein.

[0084] As used herein, the terms “prenylation,”“isoprenylation,” or “lipidation” refers to the addition of hydrophobic molecules to a protein. Protein prenylation involves the transfer of either a farnesyl or a geranyl-geranyl moiety to C-terminal cysteine(s) of the target protein. As used herein, the terms “deprenylation,”“desoprenylation,” or “delipidation” refers to the removal of hydrophobic molecules from a protein.

[0085] As used herein, “myristoylation” refers to the PTM process wherein myristoyl group (derived from myristic acid) is covalently attached via an amide bond to the alpha-amino group of an N-terminal amino acid of a polypeptide. It is more common on glycine residues but also occurs on other amino acids. Myristoylation occurs post-translationally, for example when previously internal glycine residues become exposed by caspase cleavage during apoptosis. As used herein, “demyristoylation” refers to the PTM alteration wherein myristoyl group (derived from myristic acid) is removed from the alpha-amino group of an N-terminal amino acid of a polypeptide.

[0086] As used herein, “S-palmitoylation” refers to the covalent attachment of fatty acids, such as palmitic acid, to cysteine residues of proteins. As used herein, “de-S-palmitoylation” refers to the removal of fatty acids, such as palmitic acid, to cysteine residues from proteins.

[0087] As used herein, “tyrosine sulfation” is a PTM where a sulfate group is added to a tyrosine residue of a protein molecule. As used herein, “tyrosine desulfation” is a PTM alteration where a sulfate group is removed from a tyrosine residue of a protein molecule.

[0088] As used herein, “deamidation” refers to the chemical reaction in which an amide functional group is removed from a protein. The reaction damages the amide-containing side chains of the amino acids asparagine and glutamine.

[0089] As used herein, “formylation” is a type of PTM in which a formyl group is added to the N-terminus of a protein. As used herein, “deformylation” is a type of PTM alteration in which a formyl group is removed from the N-terminus of a protein.

[0090] As used herein, “carboxylation” is a PTM in which a carboxylic acid group is added to glutamate residues in proteins. It occurs primarily in proteins involved in the blood clotting cascade, specifically factors II, VII, IX, and X, protein C, and protein S, and also in some bone proteins. As used herein, “decarboxylation” is a PTM alteration in which a carboxylic acid group is removed from glutamate residues in proteins.

[0091] In some embodiments of the present invention, the PTM reaction is a modification of proteins with a ubiquitin-like modifier selected from the group consisting of ISG15, UCRP, FUB1, NEDD8, FAT10, SUMO-1, SUMO-2, SUMO-3, Apg8, Apg12, Urm1, UBL5, and Ufm1 (see Table 1 for further description). In other embodiments of the present invention, the PTM reaction is one of ubiquitination, sumoylation, and neddylation.

[0092] The methods described herein can be used to detect changes both in PTM enzyme activity and its cognate protein targets in a patient through the analysis of a patient sample, such as plasma, CSF, or from an extract prepared from biopsy tissue. There is great need for a method that is capable of rapidly detecting biomarkers of diseases such as Alzheimer's disease or cancer in a patient sample, and to distinguish the disease from the normal state. Detecting PTMs of a large number of proteins provides a detailed fingerprint of the PTM enzymes released from tissues during disease.

[0093] In some embodiments, the functional cell extract for use in the methods described herein is obtained from a biological sample. As used herein, a “biological sample” includes, but is not limited to, saliva, blood, umbilical cord blood, serum, plasma, urine, cerebrospinal fluid (CSF), chorionic villus, lymph fluid, placenta, breast milk, nipple aspirates, pleural fluid, mucus, semen, vaginal secretions, any cell sample (heterogenous or homogenous), any solid tissue, a tumor, amniotic fluid, and a tissue culture sample. Tissue samples include but are not limited to, skin tissue, lung tissue, adipose tissue, connective tissue, sub-epithelial tissue, epithelial tissue, liver tissue, kidney tissue, uterine tissue, respiratory tissues, gastrointestinal tissue, and genitourinary tract tissue. In some embodiments, the sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary or metastatic tumor, or a cell block from pleural fluid. In addition, fine needle aspirate samples can be used. A cell sample includes, for example, a population of cells obtained from a single-cell suspension of a tissue, for example, spleen, lymph node, or thymus. In some embodiments, a cell sample can be a heterogenous population of cells, such as the population of immune cells found in the spleen. In other embodiments, a cell sample refers to a purified population of cells, such as purified T or B cells isolated from lymph node tissue by methods known to one of skill in the art. In other embodiments, the functional cell extract can be directly prepared from a tissue or tumor by homogenization of the tissue or tumor. In some embodiments, the tumor sample refers to a biopsy of a tumor. Regarding extracellular fluids, such as interstitial fluids, lymph, CSF, blood, serum, plasma, urine, saliva, umbilical cord blood, amniotic fluid, breast milk, mucus, semen, and vaginal secretions, it is still unclear how certain intracellular proteins are deposited in such extracellular fluids, though they are expected to result from cellular turnover; nevertheless, many examples of intracellular proteins in such fluids are known. For example, it is known that cytoskeletal proteins such as tau and post-translationally modified forms thereof (phospho-tau) can be readily detected in CSF from patients suffering from Alzheimer's disease. Prior to the present invention, however, it was unknown whether functional PTM enzymes are present in extracellular fluid samples such as CSF and plasma and could be used to modify target proteins. Thus, the invention now provides a means to assay PTM enzyme activities in samples that were previously not used for such analysis.

[0094] In other embodiments, the methods described herein are useful for assaying PTM or PTM alterations of frozen or cryopreserved biological samples. Biological samples that can be frozen or cryopreserved include, but are not limited to, any of the biological samples described herein. Previously, the methods used to assay PTM or PTM alterations were limited to the use of fresh biological samples, i.e., those taken from a subject and processed immediately, or those extracts obtained from an in vivo source and processed ex vivo (i.e., isolated cells). As used herein, “cryopreservation” refers to the process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as, 77 K or −196° C. (the boiling point of liquid nitrogen). For example, machines can be used that freeze biological samples, to be used in the methods described herein, using programmable steps, or controlled rates, before it is deep frozen, or by cryopreserving such samples in liquid nitrogen. Such machines can be used for freezing any of the biological samples described herein, including blood products, embryo, sperm, stem cells, and general tissues. Freezing must be regulated carefully to preserve the integrity of the biological sample, and lethal intracellular freezing can be avoided, for example, if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. That rate differs between cells of differing size and water permeability: a typical cooling rate around 1° C. / minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide (DMSO), but the rate is not a universal optimum. In some embodiments, vitrification can be performed to prepare the cryopreserved biological sample. In clinical cryopreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. Cryoprotectants lower the freezing temperature and increase the viscosity of the biological sample, such that instead of crystallizing, the syrupy solution turns into an amorphous ice, i.e., it vitrifies. Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid drop in temperature (megakelvins per second). Many solutes do both, but larger molecules generally have larger effect, particularly on viscosity. Rapid cooling also promotes vitrification. In established methods of cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and depress freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as dimethyl sulfoxide, a common cryoprotectant, are often toxic in high concentration. One of the difficult compromises faced in vitrifying cryopreservation is limiting the damage produced by the cryoprotectant itself. In general, cryopreservation is easier for thin samples and small clumps of individual cells, because these can be cooled more quickly and so require lower doses of toxic cryoprotectants. Examples of biological samples that can be cryopreserved using vitrifying cryopreservation include, but are not limited to, semen; blood and blood products such as serum and plasma; cells; stem cells; umbilical cord blood; tissue samples like tumors and histological cross sections; oocytes; 2, 4, or 8 cell embryos; and ovarian tissue. Cryoprotectant media may be, for example, supplemented with either egg yolk or soy lecithin.

[0095] The ability to use frozen or cryopreserved biological samples provides a significant and useful improvement over the standard biochemical methods used to detect PTM or PTM alterations, as such samples can be assayed long after they are obtained, and can be used to make comparisons between samples obtained at different timepoints, and from different locations. Further, if multiple biological replicates of these samples are prepared prior to the freezing or cryopreservation, a frozen or cryopreserved biological sample can be assayed multiple times. For example, the effect of a drug or treatment on PTM and PTM alterations can be assayed using cryopreserved samples taken at different timepoints from a subject being treated for a disorder. Also, cryopreserved samples can be used to compare PTM or PTM alterations between biological samples, such as a tumor biopsy, obtained from different subjects at different locations, to determine whether one or more PTM or PTM alterations or patterns of PTM or PTM alterations are shared between the same types of tumors in different subjects.

[0096] As used herein, the term “functional extract” refers to the extract of a biological sample, either in its entirety (i.e., not diluted) or any unfractionated portion or volume portion thereof, or any dilution or concentrations thereof. The term “functional extract” also includes an extracellular fluid sample obtained from a patient, applied undiluted, diluted or concentrated, in its entirety or as any mass portion or volume portion thereof. Preferably, the functional extract is not subjected to a protein purification process prior to use in a PTM or PTM alteration reaction on a solid state array, such as a protein microarray. The extract as used for a PTM or PTM alteration reaction can be supplemented with any reagent, including salts, buffers, gases, substrates, enzymes, inhibitors, etc., as desired or as appropriate for the particular PTM or PTM alteration reaction being performed.

[0097] A functional cell extract derived from a biological sample for use in the methods described herein to detect PTMs and PTM alterations can be an undiluted or concentrated extract. Accordingly, in some embodiments, the functional cell extract is not diluted prior to contacting with a solid state array. In some embodiments, functional cell extracts of patient samples or biological samples are preferably maintained at a protein concentration approaching that of in the body of the subject, so that protein-protein interactions that might affect activity are retained in the extract. In other embodiments, the functional cell extract is concentrated prior to contacting with a solid state array. In some such embodiments, the functional cell extract is highly concentrated prior to contacting with a solid state array. In such embodiments where a concentrated functional cell extract is used, the method of concentration does not involve protein purification or protein removal from the extract, but rather removal of extra cellular fluid or buffers used to isolate and prepare the cellular extract. For example, when a cell lysis solution is used to lyse a biological sample for use in the methods described herein, methods of protein concentration known to those of skill in the art can be used to concentrate the sample to form the functional cell extract prior to contacting with a solid state array for detection of a PTM or PTM alteration reaction in the extract. Non-limiting examples of methods to concentrate a functional cell extract include membrane filtering (microfiltration and ultrafiltration techniques), the use of high-speed vacuums, membrane dialysis, and TCA precipitation.

[0098] Highly concentrated cellular extracts have been shown to have demonstrable function. Such cellular extracts from Xenopus and from somatic cells that demonstrated a function specified for a particular phase of the cell cycle have allowed for the recapitulation of complex events, such as the ordered degradation of mitotic substrates (1). Also, in recent years, these systems have been employed for an in vitro expression cloning (IVEC) screening approach (2) and were used successfully to identify proteins that undergo mitosis-specific degradation (3, 4), apoptotic protease substrates (5), protein kinase substrates (5), and other binding interactions (6).

[0099] A functional cell extract derived from a biological sample for use in the methods described herein to detect PTMs and PTM alterations is essentially devoid of detergents or surfactants, as well as toxins or substances that could inhibit the biological function of components of the extract, e.g., enzymes and co-factors involved in PTM reactions, or that could denature or alter the protein targets in the microarray. In contrast to the methods described in US2008 / 0138836, where a commercial buffer containing three detergents are used to prepare an extract, the methods described herein allow an artisan to use a detergent-free or essentially devoid of detergents functional cell extract for detecting PTM or PTM alterations on a solid state array. Accordingly, in some embodiments, an essentially detergent-free functional cell extract is contacted with a solid state array for detecting a PTM or PTM alteration. In some embodiments, a functional cell extract is prepared from a biological sample using one or more detergent-free or essentially detergent-free solutions. In some embodiments, the functional cell extract is detergent-free. Negligible amounts of detergents, toxins, or other factors that do not affect PTM activity may be present.

[0100] A non-limiting example of a method for preparing a functional extract from a cell sample is to use a gentle, minimally diluting method such as one or more cycles of freeze-thaw, optionally combined with mildly hypotonic lysis of cells that may be present in the sample. The amount of sample material used to prepare the extract will depend on the scale of the experiment, such as the number and size of the microarrays used, but generally at least one million cells or at least an amount of tissue or bodily fluid equivalent to 50 microliters of an undiluted lysed tissue sample or cell extract, or at least about 20 μl of a bodily fluid such as plasma or cerebrospinal fluid is sufficient for preparing an extract to cover a single 1×3 inch microarray.

[0101] In order to prepare a functional extract from a cell sample, cells are first harvested using standard techniques for collecting cells, e.g., from culture or from a specimen obtained from a patient. Such techniques can include, for example, single-cell suspension preparation, tissue homogenization, treatment of tissue or cell culture with trypsin, collagenase, or other enzymes, passage through a needle, sonication, or separation by centrifugation or passage through a column, such as an affinity column. In other embodiments, purified cells can be obtained using methods and techniques known to skilled artisan for cell purification and isolation, such as magnetic bead isolation using columns, or via flow cytometric sorting techniques. Cells can be swelled in a buffer such as 25 mM HEPES, pH 7.5, containing 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, optionally containing a preferred mixture of protease inhibitors, such as COMPLETE™ protease inhibitors (Roche). In some embodiments, in order to concentrated the functional cell extract, the ratio of lysis or homogenization solution preferably is kept to a minimum, e.g., similar to or less than the volume of cells being extracted, in order to minimize the dilution of extracted material. In some embodiments, a ratio of about 0.5 to 1 volume of lysis solution to cell volume can be used to concentrate the functional cell extract. In some embodiments, preferably 0.8 volumes or less of lysis solution is used for each volume of cells to be disrupted to form the concentrated functional cell extract. After homogenization, the crude cell extract can be treated to remove membranes and whole or fragmented cells, such as by centrifugation.

[0102] In some embodiments, the functional cell extract for use in the methods described herein is derived from one or more specified cellular compartments. In such embodiments, the functional cell extract derived from one or more specified cellular compartments can also be concentrated prior to contact with a solid state array. In one embodiment, the cellular compartment is nucleus. In another embodiment, the cellular compartment is cytosol. In another embodiment, the cellular compartment is mitochondria. In one embodiment, the cellular compartments are nucleus and cytosol. In one embodiment, the cellular compartments are nucleus and mitochondria. In one embodiment, the cellular compartments are cytosol and mitochondria. In some embodiments, the functional cell extract for use in the methods described herein lacks one or more specified cellular compartments. In one embodiment, the functional extract lacks nucleus. In one embodiment, the functional extract lacks cytosol. In one embodiment, the functional extract lacks mitochondria. Functional extracts can be made from these different cellular compartments according to published protocols known to one of skill in the art.

[0103] Functional extracts can be prepared from any suitable source of cells, tissue, or biological fluid that can be obtained from a patient or subject. The patient or subject can be a human or a non-human animal. The terms “subject”, “patient” and “individual” are used interchangeably herein, and refer to an animal, for example a human, from whom the biological sample can be obtained from. For treatment of disease states which are specific for a specific animal such as a human subject, the term “subject” refers to that specific animal. The terms “non-human animals” and “non-human mammals” are used interchangeably herein, and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g. dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like are also encompassed in the term subject. Sources of cells or tissue for extraction can include, for example, a biopsy specimen, such as a tumor or suspected tumor, serum, plasma, cerebrospinal fluid, saliva, urine. Non-cellular (e.g., bodily fluid, interstitial fluid) samples usually contain intracellular content that is sufficient for analysis; such content may be derived, for example, from directed secretion from cells, from inflammation, or tissue damage. In other embodiments, a non-cellular biological sample comprises the media obtained from tissue culture samples.

[0104] A functional extract can be supplemented with one or more substances to aid in the analysis of a specific post-translational state or a specific PTM enzyme or PTM modifying enzyme activity. For example, an extract can be supplemented with a reagent, a substrate, an enzyme, an enzyme inhibitor, a drug, an antibody, or any mixture thereof. Alternatively, an extract can be depleted using antibodies directed to a chosen protein, protein complex, or modified protein. An extract lacking a particular protein component also can be prepared from knockout or knockdown cells. In some embodiments of the methods described herein, an additional cellular energy source in the form of, for example, ATP is provided to the functional cell extract. In one embodiment, a biochemical energy source such as ATP plus an ATP regenerating system is added to the extract or fluid to establish a reaction on the microarray. A high concentration of creatine phosphate (e.g. 150 mM) is a suitable ATP-regenerating system. Creatine phosphokinase can also be added in addition to creatine phosphate, but may be omitted if sufficiently present in the extract or fluid. Preferably, a substrate for a PTM enzyme, such as ubiquitin, is also added to the extract or fluid to establish a specific PTM reaction.

[0105] For some PTM reactions (e.g., ubiquitination, requiring E1, E2, and E3 enzymes), more than one enzyme is necessary to carry out the reaction, and while one or more enzyme is supplied by the extract or fluid sample, one or more other enzymes required for optimal activity may be limited or missing. In such cases the missing or limited enzyme or enzymes can be added to the extract or fluid to establish an optimal PTM reaction or PTM alteration reaction. A further useful strategy is to add to the extract an inhibitor of an enzyme that inhibits a particular type of PTM or PTM alteration. Examples include methyl-ubiquitin and dominant-negative E2 enzymes for ubiquitination or sumoylation. An exemplary list of enzymes that might be added to supplement a PTM reaction is provided in Table 1. One skilled in the art can readily identify additional enzymes and enzyme combinations based on existing or acquired knowledge of PTM pathways and reactions. The methods of the invention do not depend on specific combination of components.

[0106] TABLE 1UbiquitinUbiquitin-LikeSequenceE1-E2-E3 Conjugating EnzymesModifierHomology (%)Deconjugating Enzyme (DCE)SubstratesFunctionsISG15 (UCRP)29, 27E1: UBE1L; E2: UBCH8PLCγ1, JAK1, STAT1,Positive regulator of (2 ubiquitins)ERK1 / 2, serpin 2aIFN-relatedimmune response, potentiallyinvolved in cell growth anddifferentiationFUB1 (MNSFβ)37NATCR-α-like protein, Bcl-GNegative regulator of leukocyteactivation and proliferationNEDD8 (Rub1)58E1: APPBP1-UBA3; E2: UBC12; E3:cullins, p53, Mdm2, Positive regulator of Roc1, Mdm2; DCE: DEN1 / NEDP1,synphilin-1 ubiquitin E3s; directs to UCH-L1, UCH-L3, USP21, COP9proteasomal degradationFAT10 (229, 36NAMAD2Cell cycle checkpoint ubiquitins)for spindle assembly, directs to proteasomaldegradationSUMO-1 18E1: SAE-1 / -2 (AOS1-UBA2); E2:Glut1, Glut4, c-Jun, Control of protein (SMT3C,UBC9; E3: RanBP2, Pc2, PIAS1κBα, p53, Mdm2, SOD-1, stability, function,GMP1, UBL1)superfamily; DCE: SENP-1 and-2RXRα, NEMO,and localization, (Ulp-1 and-2), SUSP4PML, Sam68, RanGAP1,antagonist toRanBP2, ADAR1, PCNA,ubiquitin, overlap Drp1, STAT-1, Sp3, with SUMO-2 / -3thymine-DNA glycosylase,topoisomerase IISUMO-2 16E1: SAE-1 / -2; E2: UBC9; DCE:RanGAP1, C / EBPβ1,Transcription regulation, (SMT3B);SENP-3 and-5topoisomerase II, thymine-cell cycle progressionSUMO-3 DNA glycosylase(SMT3A)Apg 810E1: Apg7; E2: Apg3; DCE: Apg4PhosphatidylethanolamineAutophagy, cytoplasm-to-vacuole targetingApg 1217E1: Apg7; E2: Apg10Apg 5Autophagy, cytoplasm-to-vacuole targetingUrm112E1: Uba4Ahp1Potential role in oxidative stress responseUBL5 (Hub1)25NACLK4, Snu66, Sph1, Hbt1Pre-mRNA splicing, appetiteregulationUfm116E1: Uba5; E2: Ufc1NAPotential role in endoplasmic stress response

[0107] Small molecule inhibitors may also be used in a PTM reaction. Additionally, adenosine 5′-(gamma-thio)triphosphate can be added as an inhibitor of ATP-dependent processes in an extract. Also, certain proteases can be inhibited, removed, or supplemented into the reaction in order to check their effect or to find specific targets.

[0108] Any solid state array can be used for the methods described herein. A “solid state array,” as used herein, refers to any combination of one or more target proteins or peptides attached to a solid support. Such a support can be a microchip, a bead, a glass slide, or any other support suitable for arraying a target protein or peptide. An array for use in the invention also can be fabricated in any desired format or dimensions and with any desired number of target proteins, as long as the position of each target protein is known and the target can be identified by its position within the array. Accordingly, in some embodiments, the solid state array for use in the methods described herein includes protein arrays on microchips, ELISA plates with immobilized proteins attached on the plates, protein-coated beads, and microfluidic chips coated with desired proteins. In some embodiments of this aspect, 2-10 PTM or PTM alterations are identified simultaneously. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more PTM or PTM alterations can be screened in one assay with suitable detection methods, such as labeled antibodies. In some embodiments, the multiple PTMs, PTM alterations, or combinations thereof are detected in parallel. In some embodiments, multiple PTMs, PTM alterations, or combinations thereof are detected sequentially. In such embodiments, the first PTM may affect the second PTM. Such sequential identification of PTM or PTM alteration allows one to determine PTM pathways and screen for different agents affecting various parts of the PTM or PTM alteration pathway. In some embodiments, multiplex analysis of 10-15, 10-100 PTM and / or PTM alteration reactions can be performed.

[0109] A protein microarray for use in the methods of the invention can be selected from commercially available or in-house microarrays. The array has a substrate upon which proteins are deposited in a two-dimensional array (i.e., an ordered plurality of proteins), such that each position in the array contains a single type of known protein whose PTM or PTM alteration can be investigated. The substrate of the array can be made of a material such as a glass slide, to which protein molecules are covalently or non-covalently bound. Optionally, glass can be coated with nitrocellulose or derivatized with expoxy or amino groups to provide desirable surface properties, to reduce non-specific binding, or to provide attachment points for proteins. An example of a commercially available protein microarray suitable for use in the invention is the PROTO-ARRAY® Human Protein Microarray from Invitrogen, which contains over 8000 human proteins. Other commercially available or user prepared arrays or microarrays can be used as well. In some embodiments of the methods described herein, the array comprises at least one protein, protein fragment, or peptide attached to the array with a C-terminal or N-terminal tag. Selected proteins, for example recombinant proteins that are N-terminally or C-terminally tagged and purified, can be used to prepare any desired protein microarray for use in the invention. In other embodiments, the array comprises at least one protein, protein fragment or peptide attached to the array without an added tag or moiety to facilitate binding to the solid support.

[0110] A protein array for use with the invention can have at least 2, 5, 10, 100, 1000, 8000, 10,000, 30,000, or 100,000 or more individual protein spots or wells in the array, in addition to which other locations can be added to the array for controls or background determination, or other purposes as desired. The individual proteins in the array can be all distinct, or the proteins at some positions can be identical to proteins at other positions, or can be variants (e.g., sequence mutants or differently modified versions) of proteins at other positions.

[0111] An alternative to using a protein microarray for detection is to use an array constructed from a microtiter plate or any similar container having a plurality of wells. Individual target proteins can be added to individual wells at known locations for carrying out the PTM or PTM alteration reaction and detection. It is only necessary to retain the proteins at their respective locations throughout the reaction, washing, and detection steps. For example, recombinant proteins bearing a tag, such as a GST, FLAG, or myc tag, can be coupled to glass beads that are deposited at specific locations in a microtiter plate. The beads can be retained in the wells during solution exchange, and offer the possibility to uncouple and release the modified proteins for further study, e.g., by mass spectrometry. In other embodiments, the recombinant proteins are directly deposited at specific locations in a microtiter plate, and binding is mediated by the properties of the microtiter plate. For example, untreated and irradiated polystyrene microtiter plates permit hydrophobic and hydrophilic interactions between the polystyrene and the protein being deposited.

[0112] Another alternative to using a protein microarray for detection is to use a solid state array comprising beads to which the protein targets of the PTM or PTM alteration are attached, such as a multiplex bead assay. For example, in some embodiments, protein targets of a PTM or PTM alteration are attached to beads of different sizes or colors (emission spectra) in a multiplex bead based assay. In such embodiments, a plurality of beads of different sizes is coated with different protein targets of a PTM or PTM alteration, wherein each bead of a specific size is conjugated to a specific protein target. Accordingly, each bead can be differentiated by its unique light scatter characteristics. A biological sample, such as a blood sample, to be assayed for the presence of at least one PTM or PTM alteration is then contacted with a plurality of beads of different sizes having different protein targets, thus allowing the PTM or PTM alteration to occur on one or more protein targets attached to specific beads.

[0113] In some embodiments of this aspect, such bead-based technology can be employed wherein bead populations are identified by one type of fluorescence, while the PTM or PTM alteration of the protein target on the bead is generated by one or more detection reagents carrying a second type of fluorescent signal, thus creating a bead set specific for detecting a plurality of PTM or PTM alteration. In preferred embodiments, the distinguishable bead populations are prepared by staining the beads with two or more fluorescent dyes at various ratios. Each bead having a specific ratio of the two or more fluorescent dyes is conjugated to a specific protein target, thus assigning each bead-protein target a unique fluorescent signature. The immunoassay signal is generated by detection reagents, coupled to a third type of fluorescent dye. A biological sample to be assayed for the presence of at least PTM or PTM alteration is then contacted with the plurality of beads with unique fluorescent signatures and protein target specificity, forming a PTM or PTM alteration on specific beads having the protein target of that PTM or PTM alteration. The presence of each of the at least one PTM or PTM alteration can be ascertained by flow cytometric analyses on the bead bound-target proteins. For example, in some embodiments, beads are dyed with fluorochromes having different fluorescence intensities. In some embodiments, the beads are 7.5 μm in diameter. In some embodiments, the fluorescent dye incorporated in the beads fluoresces strongly at 650 nm upon excitation with an argon laser. Each bead population of a given fluorescence intensity represents a discrete population for constructing an immunoassay for a single protein target. Each bead population having a given fluorescence intensity upon excitation is covalently coupled with a specific protein target. For example, a target of an E1 ligase. These target protein-bound bead populations, each of which are unique in their fluorescence emission intensity, serve as targets for specific PTM or PTM alteration enzymes present in a biological sample.

[0114] Accordingly, as defined herein a “capture bead” is a bead having a unique fluorescence emission intensity conjugated to a specific target protein. When these capture beads specific for different target proteins are used as a mixture, different PTM or PTM alterations, can be simultaneously measured within a given sample. In some embodiments, detection is mediated by the binding of a specific detection antibody, for example, an antibody that detects any PTM or PTM alteration present in a sample, that is directly conjugated with a fluorescent tag, such as phycoerythrin (PE), to each of the modified protein targets present after contacting with the biological sample, thus providing a second fluorescent signal for each capture bead. The fluorescent signal is proportional to the concentration of the biomarker in the sample. Separately established calibration curves can be used to determine the degree of PTM or PTM alteration in the test sample, using dedicated analysis software, such as CBA software.

[0115] The data collected using a flow cytometer includes information about the physical and spectral parameters of the beads, such as size and the fluorescence emission characteristics of each bead population. These fluorescence emission characteristics include the fluorescent emission of the dyed beads, and the potential fluorescent emissions of the detection fluorochrome (for example, phycoerythrin). When samples are analyzed using a flow cytometer in conjunction with a typical data acquisition and analysis package (for e.g., BD CellQuest™ software), a list-mode data file is saved using a flow cytometry standard file format, FCS. The data stored in the FCS files can be reanalyzed to determine the median fluorescence intensities (MFI) of the various bead populations, defined by their unique physical and spectral characteristics, to then compare reference samples with unknowns. The PTM or PTM alterations being assayed within individual samples can then be calculated from calibration curves generated by serial dilutions of standard solutions having known PTM or PTM alterations. An automated or semiautomated analysis method can be used for rapid reanalysis of the data stored in each FCS file. For example, BD CBA Software is written in the MICROSOFT® Excel Visual Basic for Applications (VBA) programming language. The CBA Software can recognize FCS 2.0 and 3.0 format data files and automates the identification of CBA bead populations and the determination of detector fluorochrome MFI values for each bead population within the data file for a single sample. Using this data analysis function of the CBA Software for multiple standard files, the MFI values for standards are then determined and plotted. From the plotted standard curve and complex mathematical interpolation, values for unknown samples can be rapidly determined in comparison to known standards using the software.

[0116] A functional extract is contacted with a solid state array, such as a protein microarray, usually by depositing an aliquot or portion of the extract, optionally after dilution or supplementation with a reagent or buffer, which may include an energy source, such as ATP and / or one or more enzymes that take part in the PTM or PTM alteration reaction, onto the surface of the microarray where proteins are deposited. Alternatively, supplements can be added after the extract is deposited onto the microarray. Once contacted with the microarray, the extract can be incubated under any desired conditions, such as at room temperature or another temperature (e.g., 30 or 37° C.), suitable to promote the protein-protein interactions and enzyme reactions necessary to allow a PTM state to be established. Generally, the incubation will last for a period ranging from several minutes to hours. The incubation conditions should be sufficient to permit a steady state level for the particular PTM reaction under consideration to be established.

[0117] One method of the invention involves detection and analysis of altered states of PTM in one or more proteins in a biological sample from a patient compared to a biological sample from a control patient, or control data, or data obtained from the same patient at an earlier time. A state of PTM can be altered, for example, if there is a change in the average number of a given chemical group attached per protein molecule, if there is a change in the type of chemical group or groups attached per protein molecule, or if there is a different mixture of protein molecules having distinct modification patterns in a patient sample. Alteration of a PTM state of a protein includes going from an unmodified protein to a modified one and vice-versa, as well as changes in the number or type of chemical moieties added to the protein.

[0118] Thus, one embodiment of the invention is a method of identifying an altered PTM state of a protein in a patient. The method includes the steps of (i) contacting a functional extract of a sample from the patient with a protein microarray containing proteins that are representative of proteins in the patient; (ii) establishing a specific PTM reaction on the microarray, whereby the reaction results in a PTM of one or more proteins in the microarray through the activity of one or more enzymes present in the extract; (iii) determining the level of PTM of proteins in the microarray; and (iv) comparing the levels of PTM with PTM levels of corresponding proteins in a control sample to identify altered PTM states of one or more proteins in the patient.

[0119] A specific PTM reaction can be established on an array by adding a substrate (e.g., ubiquitin) to the extract or fluid sample that is required for a single PTM reaction. An assay also can be rendered specific for a single PTM reaction by the use of an antibody that detects only one specific PTM state. Methods according to the invention can be addressed to either a single specific PTM reaction at a time or more than one specific PTM reactions performed simultaneously in the same reaction mixture (multiplex format).

[0120] The particular target proteins in the microarray can be selected so as to be representative of the proteins available in the patient. For example, the microarray can include a large number of human proteins if the patient is a human patient. In one embodiment, the proteins in the microarray are initially in an unmodified state, such as that obtained by expressing the proteins in a recombinant expression system that does not modify the proteins. In another embodiment, the proteins in the microarray have various states of PTM; such proteins can be further modified by a functional extract, providing differential modification signals. Alternatively, in another embodiment the target proteins in the array can be biochemically stripped of certain PTMs prior to exposure to the functional extract for analysis. During the step of contacting the functional extract with the microarray, one or more proteins in the array will become post-translationally modified by the enzymes, cofactors, and substrates in the extract.

[0121] Following an appropriate incubation period, the cell extract can be washed off the microarray by standard techniques, including spin drying, centrifugation, or blowing a stream of gas (e.g., air or nitrogen) over the surface of the microarray followed by application of a buffer solution to the microarray. The washing step can be repeated as needed to remove components from the cell extract from the microarray, leaving the modified target proteins attached to the microarray for subsequent detection. A suitable washing solution is a Tris buffered saline solution (TBS), optionally supplemented with one or more detergents (e.g., 0.05% Tween, or for more stringent conditions 0.5% SDS) to dissociate non-specifically bound proteins from the proteins in the array.

[0122] After the cell extract has been removed, the next step is to determine the level of PTM of individual proteins in the microarray. This can be accomplished, for example, using an antibody that specifically binds all proteins having a specific type of modification. Many such antibodies are commercially available, such as Anti-Polyubiquitin (BioMol), anti-ubiquitin (with specific linkages, Cell Signaling), anti-sumo1 (Cell Signaling, BioMol), anti-sumo2 / 3 (Cell Signaling, Biomol), anti-NEDD8 (Biomol, MB1, Sigma), anti-APG8 (Boston Biochem), anti-FAT10 (Boston Biochem), and anti-UFM1 (Boston Biochem). Examples of commercially available antibodies that can be used to specifically detect different PTM and PTM alteration states are listed in Table 2.

[0123] TABLE 2PTM Detected / AntibodyCatalog NumberCompanyUbiquitin monoclonal mouse monoclonalAB-001CellSignalingSUMO2 polyclonal mouse polyclonalAB-S80CellSignalingSUMO2 monoclonal mouse monoclonalAB-S81CellSignalingSUMO3 MaxPab polyclonal mouse polyclonalAB-S90CellSignalingSUMO3 polyclonal mouse polyclonalAB-S91CellSignalingSUMO3 monoclonal mouse monoclonalAB-S92CellSignalingSUMO3 monoclonal mouse monoclonalAB-S93CellSignalingSUMO4 MaxPab polyclonal mouse polyclonalAB-S95CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S96CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S97CellSignalingAnti-NEDD8 rabbit polyclonalA-812CellSignalingAnti-UBE1L (E1) rabbit polyclonalA-306CellSignalingAnti-ISG15 rabbit polyclonalA-600CellSignalingUBE2L6 (UbcH8) MaxPab polyclonal mouseAB-242CellpolyclonalSignalingUBE2L6 polyclonal mouse polyclonalAB-243CellSignalingUBE2L6 (UbcH8) monoclonal mouse monoclonalAB-244CellUbiquitin monoclonal mouse monoclonalAB-001CellSignalingSUMO2 polyclonal mouse polyclonalAB-S80CellSignalingSUMO2 monoclonal mouse monoclonalAB-S81CellSignalingSUMO3 MaxPab polyclonal mouse polyclonalAB-S90CellSignalingSUMO3 polyclonal mouse polyclonalAB-S91CellSignalingSUMO3 monoclonal mouse monoclonalAB-S92CellSignalingSUMO3 monoclonal mouse monoclonalAB-S93CellSignalingSUMO4 MaxPab polyclonal mouse polyclonalAB-S95CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S96CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S97CellSignalingAnti-NEDD8 rabbit polyclonalA-812CellSignalingAnti-UBE1L (E1) rabbit polyclonalA-306CellSignalingAnti-ISG15 rabbit polyclonalA-600CellSignalingUBE2L6 (UbcH8) MaxPab polyclonal mouseAB-242CellpolyclonalSignalingUBE2L6 polyclonal mouse polyclonalAB-243CellSignalingSignalingISG15 MaxPab polyclonal rabbit polyclonalAB-I10CellSignalingISG15 monoclonal clonal mouse monoclonalAB-I11CellSignalingAnti-UFM1 rabbit polyclonalA-500CellSignalingAPG3 polyclonal mouse recombinantAB-A10APG3CellSignalingAPG3 monoclonal mouse monoclonalAB-A11APG3CellSignalingAPG4B polyclonal rabbit polyclonalAB-A20APG4BCellSignalingAPG4C MaxPab polyclonal mouse polyclonalAB-A21APG4CCellUbiquitin monoclonal mouse monoclonalAB-001CellSignalingSUMO2 polyclonal mouse polyclonalAB-S80CellSignalingSUMO2 monoclonal mouse monoclonalAB-S81CellSignalingSUMO3 MaxPab polyclonal mouse polyclonalAB-S90CellSignalingSUMO3 polyclonal mouse polyclonalAB-S91CellSignalingSUMO3 monoclonal mouse monoclonalAB-S92CellSignalingSUMO3 monoclonal mouse monoclonalAB-S93CellSignalingSUMO4 MaxPab polyclonal mouse polyclonalAB-S95CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S96CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S97CellSignalingAnti-NEDD8 rabbit polyclonalA-812CellSignalingAnti-UBE1L (E1) rabbit polyclonalA-306CellSignalingAnti-ISG15 rabbit polyclonalA-600CellSignalingUBE2L6 (UbcH8) MaxPab polyclonal mouseAB-242CellpolyclonalSignalingUBE2L6 polyclonal mouse polyclonalAB-243CellSignalingSignalingAPG4C polyclonal rabbit polyclonalAB-A22APG4CCellSignalingAPG5 monoclonal mouse monoclonalAB-A25APG5CellSignalingAPG7 MaxPab polyclonal mouse polyclonalAB-A30APG7CellSignalingAPG7 polyclonal rabbit polyclonalAB-A31APG7CellSignalingAPG9A polyclonal rabbit polyclonalAB-A40APG9CellSignalingAPG10 polyclonal rabbit polyclonalAB-A50APG10CellSignalingAPG10 polyclonal rabbit polyclonalAB-A51APG10CellUbiquitin monoclonal mouse monoclonalAB-001CellSignalingSUMO2 polyclonal mouse polyclonalAB-S80CellSignalingSUMO2 monoclonal mouse monoclonalAB-S81CellSignalingSUMO3 MaxPab polyclonal mouse polyclonalAB-S90CellSignalingSUMO3 polyclonal mouse polyclonalAB-S91CellSignalingSUMO3 monoclonal mouse monoclonalAB-S92CellSignalingSUMO3 monoclonal mouse monoclonalAB-S93CellSignalingSUMO4 MaxPab polyclonal mouse polyclonalAB-S95CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S96CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S97CellSignalingAnti-NEDD8 rabbit polyclonalA-812CellSignalingAnti-UBE1L (E1) rabbit polyclonalA-306CellSignalingAnti-ISG15 rabbit polyclonalA-600CellSignalingUBE2L6 (UbcH8) MaxPab polyclonal mouseAB-242CellpolyclonalSignalingUBE2L6 polyclonal mouse polyclonalAB-243CellSignalingSignalingAPG12 MaxPab polyclonal mouse polyclonalAB-A64APG12CellSignalingAPG12 polyclonal rabbit polyclonalAB-A65APG12CellSignalingAPG12 monoclonal mouse monoclonalAB-A66APG12CellSignalingURM1 polyclonal rabbit polyclonalAB-O30CellSignalingAnti Fat10 (Protein derived)PW9680-002Biomolanti-Fat10 PolyclonalPW9585-0025Biomolanti-URM1 polyclonalPW9595-0025Biomolanti-FUB1 polyclonalPW9615-0025BiomolMouse Anti-O-GlcNAc Monoclonal Antibodysc-81483Santa CruzUbiquitin monoclonal mouse monoclonalAB-001CellSignalingSUMO2 polyclonal mouse polyclonalAB-S80CellSignalingSUMO2 monoclonal mouse monoclonalAB-S81CellSignalingSUMO3 MaxPab polyclonal mouse polyclonalAB-S90CellSignalingSUMO3 polyclonal mouse polyclonalAB-S91CellSignalingSUMO3 monoclonal mouse monoclonalAB-S92CellSignalingSUMO3 monoclonal mouse monoclonalAB-S93CellSignalingSUMO4 MaxPab polyclonal mouse polyclonalAB-S95CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S96CellSignalingSUMO4 polyclonal rabbit polyclonalAB-S97CellSignalingAnti-NEDD8 rabbit polyclonalA-812CellSignalingAnti-UBE1L (E1) rabbit polyclonalA-306CellSignalingAnti-ISG15 rabbit polyclonalA-600CellSignalingUBE2L6 (UbcH8) MaxPab polyclonal mouseAB-242CellpolyclonalSignalingUBE2L6 polyclonal mouse polyclonalAB-243CellSignalingS-nitrosocysteine antibodyab50185AbcamAcetylated-Lysine Antibody#9441CellSignalingacetyl Lysine antibodyab76AbcamCitrulline polyclonal antibodyPAB0068Abnova

[0124] In order to visualize the specifically bound antibody molecules on the microarray, the unbound first antibody is first washed away and a second antibody (e.g., an anti-immunoglobulin that specifically binds the first antibody) can be added to the microarray and allowed to bind with the first antibody. The second antibody can be labeled, e.g., by conjugation to a label moiety such as a fluorescent dye, so as to generate a signal permitting detection by a microarray scanner, such as a GenePix 4000B (Molecular Devices). Preferably, the signal emitted to detect post-translationally modified proteins in the microarray is a light signal, though other signals such as radioactivity can be used as well. The scanner can detect both the amount of signal and its position within the microarray. Two or more PTMs or PTM alterations can be detected simultaneously by using a selection of different first antibodies, each binding specifically to a different protein modification and each recognized by a different second antibody, with each second antibody conjugated to a different labeling moiety (e.g., different fluorescent dyes having excitation and emission wavelengths selected to enable simultaneous detection). An alternative method is to use labeled primary antibodies specific for the PTM or PTM alterations instead of using secondary antibodies. The data can be output as an image, or as an amount of signal detected in each spot of the microarray.

[0125] An alternative method for detecting PTM of proteins in the microarray is to add the modifying moiety (e.g., a protein such as ubiquitin or sumo that is added during the PTM reaction) in a tagged form, such as a His-, GST-, or Myc-tagged moiety, and to detect the tagged molecule using a specific antibody for the tag (e.g., anti-His, anti-GST, or anti-Myc antibody. In yet another alternative method of detection, a modification moiety can be labeled with a labeling moiety such as biotin or a 35S-labeled or radioiodinated amino acid. Phosphorylation of proteins can be detected using an antibody specific for a phosphoprotein or by adding gamma-32P-ATP into the reaction. Many techniques, such as streptavidin binding or autoradiography, can be used to visualize such labeled modification moieties instead of using antibodies, or where an appropriate antibody is not available.

[0126] Yet another method of detecting modification of proteins in the microarray is to harvest the proteins from individual spots in the array and to perform biochemical analysis, e.g., by mass spectrometry, to identify the nature of the modification, such as the number and position of modified amino acids in the protein sequence. This can be accomplished, for example, by treating individual protein-containing spots with a proteolytic enzyme such as trypsin, or by using a specifically labile chemical linkage to the substrate of the array. Quantities of individual proteins in the pg to ng range can be recovered from microarray spots; such amounts are sufficient for a wide variety of biochemical analyses, including peptide mapping, amino acid sequencing, and mass spectroscopy.

[0127] In such embodiments, the modification of proteins in the microarray can be determined by mass spectrometry such as MALDI / TOF (time-of-flight), SELDI / TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS / MS, MS / MS / MS, ESI-MS / MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference in their entirety.

[0128] The terms “mass spectrometry” or “MS” as used herein refer to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z.” In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;” Wright et al., “Proteinchip surface enhanced laser desorption / ionization (SELDI) mass spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures,” Prostate Cancer and Prostatic Diseases 2: 264-76 (1999); and Merchant and Weinberger, “Recent advancements in surface-enhanced laser desorption / ionization-time of flight-mass spectrometry,” Electrophoresis 21: 1164-67 (2000), each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. Mass spectrometry methods are well known in the art and have been used to quantify and / or identify biomolecules, such as proteins and hormones (see, e.g., Li et al., (2000), Tibtech. 18:151-160; Starcevic et. al., (2003), J. Chromatography B, 792: 197-204; Kushnir M M et. al. (2006), Clin. Chem. 52:120-128; Rowley et al. (2000), Methods 20: 383-397; and Kuster and Mann (1998), Curr. Opin. Structural Biol. 8: 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al., (1993), Science, 262:89-92; Keough et al., (1999), Proc. Natl. Acad. Sci. USA. 96:7131-6; reviewed in Bergman (2000), EXS 88:133-44.

[0129] Various methods of ionization are known in the art. For examples, Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD / FI) Matrix Assisted Laser Desorption Ionisation (MALDI) and Thermospray Ionisation (TSP). In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser-desorption / ionization mass spectrometry is used to analyze the sample. Modern laser desorption / ionization mass spectrometry (“LDI-MS”) can be practiced in two main variations: matrix assisted laser desorption / ionization (“MALDI”) mass spectrometry and surface-enhanced laser desorption / ionization (“SELDI”). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

[0130] In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and / or capture reagents that selectively bind the protein modification of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser. In another variant, the surface is derivatized with molecules that bind the protein modification of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. No. 5,719,060 and WO 98 / 59361. The two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix-containing liquid to the captured analyte to provide the energy absorbing material. For additional information regarding mass spectrometers, see, e.g., Principles of Instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094. Detection and quantification of the biomarker will typically depend on the detection of signal intensity. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually, by computer analysis etc.), to determine the relative amounts of particular biomarker. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.

[0131] The methods described herein involves detection and analysis of PTMs and PTM alterations using any composition or agent that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means, thus providing a detectable signal to identify the PTM or PTM alteration. A PTM or PTM alteration can be detected using the methods described herein, for example, if there is a change in the average number of a given chemical group attached per protein molecule, if there is a change in the type of chemical group or groups attached per protein molecule, or if there is a different mixture of protein molecules having distinct modification patterns in a patient sample with respect to a control sample. Alteration of a PTM state of a protein includes going from an unmodified protein to a modified one and vice-versa, as well as changes in the number or type of chemical moieties added to the protein. A control sample or level is used herein to describe a control patient, control or reference data, or data obtained from the same patient at an earlier time. For example, in some embodiments, a control sample is a functional cell extract obtained from a biological sample obtained from a subject not suffering from the disease being examined in the test sample. In another example, a control sample is a functional cell extract obtained population of cells obtained from the same biological source that has been treated with identical media, culture condition, temperature, confluency, flask size, pH, etc., with the exception of a test agent.

[0132] Accordingly, in some embodiments, an increase in the signal from a solid-state array compared to a background or the reaction with a control is indicative of increased PTM. The terms “increased,”“increase,” or “enhance” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,”“increase,” or “enhance” mean an increase, as compared to a reference level, of at least about 10%, of at least about 15%, of at least about 20%, of at least about 25%, of at least about 30%, of at least about 35%, of at least about 40%, of at least about 45%, of at least about 50%, of at least about 55%, of at least about 6o %, of at least about 65%, of at least about 70%, of at least about 75%, of at least about 80%, of at least about 85%, of at least about 90%, of at least about 95%, or up to and including a 100%, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 6-fold, or at least about a 7-fold, or at least about a 8-fold, at least about a 9-fold, or at least about a 10-fold increase, or any increase of 10-fold or greater, as compared to a control sample or level.

[0133] In some embodiments, a decrease in the signal from a solid-state array compared to a background or the reaction with a control is indicative of a PTM alteration. The terms “decreased,”“decrease,” or “reduce” are all used herein to generally mean a decrease by a statically significant amount; for the avoidance of any doubt, the terms “decreased,”“decrease,” or “reduce” mean a decrease, as compared to a reference or control level, of at least about 10%, of at least about 15%, of at least about 20%, of at least about 25%, of at least about 30%, of at least about 35%, of at least about 40%, of at least about 45%, of at least about 50%, of at least about 55%, of at least about 6o %, of at least about 65%, of at least about 70%, of at least about 75%, of at least about 80%, of at least about 85%, of at least about 90%, of at least about 95%, or up to and including a 100%.

[0134] Preferably, the microarray includes control spots (e.g., spotted with buffer but no protein or of GST spotted on the array) distributed across the array which can be used for background subtraction or normalization. Analysis of the distribution of background signal intensities as well as the distribution of control modified protein signal intensities, taking into account the signal-to-noise ratio, will suggest an appropriate threshold level of signal intensity considered to be significant enough to represent a positive result (i.e., detection of a post-translationally modified protein).

[0135] After the level of the PTM state for one or more proteins in the solid state array, such as a microarray has been detected, alteration of this state can be identified by comparing the results for each individual protein to similar results obtained using a control sample. The control sample can be obtained from another patient, for example, or obtained from the same patient at an earlier date or from a control tissue sample obtained from another subject. A functional extract prepared from the control is used in the same method as for the test subject and applied to a second protein microarray, preferably an identical microarray to the first microarray used for the test subject, having the same proteins as the first microarray. Alternatively, comparison data can be used that have been generated using a set of patients, or data representing known or defined ratios of certain modifications. The level of a PTM state for a given protein in the first microarray (results for the test subject) is compared to the level obtained for the corresponding protein in the second microarray. Analysis of the change in state, e.g., the direction and extent of change, or the presence or absence of any change, optionally can be used to diagnose a disease or medical condition, to determine a physiological, metabolic, or developmental state, to assess the effectiveness of a drug in the patient, or to identify target proteins for treatment based on either different modification activity or different modification state.

[0136] The analysis of functional extracts using protein microarrays can also be applied to a method for identifying a PTM state of a protein. This method can be applied either to a patient sample, or to any specimen of cells or living tissue. A functional extract is prepared from the patient sample or cell or tissue specimen, as outlined above. The extract, or a portion or dilution of the extract, is contacted with a protein microarray as described earlier, and one or more proteins in the microarray becomes post-translationally modified, or a PTM becomes altered (e.g., degree of polyubiquitination) or is removed, i.e., PTM alteration. Optionally, the extract is supplemented with one or more reagents, co-factors, substrates, enzymes, or antibodies either prior to or during the step of contacting the microarray. A signal is then detected from the modified proteins in the array, such as the fluorescence signal obtained from primary and labeled secondary antibodies as described previously. The signal, preferably background subtracted, is correlated with the identity of the protein in the respective position in the microarray, which results in identification of a PTM state of a particular protein.

[0137] A method of diagnosing a disease or medical condition related to a pattern of protein PTM can be carried out using the strategies outlined above. A functional extract is prepared from a sample of a patient suspected of having a certain disease or medical condition. The extract, or a portion or dilution of the extract, optionally substituted with one or more reagents to promote and / or stabilize a particular PTM reaction, is contacted with a protein microarray. The microarray contains an ordered array of proteins corresponding to proteins in the patient. During the incubation of the extract on the microarray, one or more target proteins in the array become post-translationally modified. The extract is washed away and the modified proteins in the microarray are detected, using a strategy such as described earlier, for example, by detecting a fluorescence signal from a primary / secondary antibody pair. The pattern of signals from the microarray are measured and recorded to form a PTM data set for the patient sample. The patient data set is compared to a standard data set containing a pattern of PTM states that is characteristic or diagnostic for the disease or medical condition.

[0138] This type of diagnostic assay can be applied to a wide variety of diseases, medical conditions, and biological states. A number of diseases or conditions for which PTMs are known or suspected to play a role are summarized in Table 3. The methods of the present invention are particularly suited to diagnosing diseases or medical conditions including, but not limited to: cancer, such as breast cancer, ovarian cancer, uterine cancer, brain cancer, including astrocytoma, renal cell carcinoma, and vascular tumors of the central nervous system; neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyelotrophic lateral sclerosis, multiple sclerosis, prion diseases, neuronal intranuclear disease, Rett syndrome, and Rubenstein-Taybi syndrome; metabolic diseases, such as diabetes mellitus, diabetic ventricular dysfunction, and gaut; immune diseases, including autoimmune diseases, rheumatoid arthritis, collagen-induced arthritis, systemic lupus erythematosus, celiac disease, encephalomyelitis, and IgA neuropathy; infectious diseases, such as viral diseases; cardiovascular diseases, such as cardiac dysfunction and atherosclerosis; and biological states such as cell cycle progression, DNA damage and repair, apoptosis, the NFkB pathway, Fanconi anemia, tumorigenesis, cellular, tissue, and embryonic differentiation, and aging. PTMs that may contribute to tumorigenesis include phosphorylation, acetylation, methylation, glycosylation, prolyl isomerization, hydroxylation, oxidation, glutathionylation, and ubiquitination.

[0139] TABLE 3MODIFIEDPTMDISEASEPROTEINREFERENCETITLEUbiquitinationCancer / tumorcMyc, HectH9 (E333The ubiquitin ligaseligase)HectH9 regulatestranscriptional activationby Myc and is essential fortumor cell proliferationUbiquitinationCancer / tumor, BreastBRCA1 (E3 ligase)34Ubiquitination andand ovarian cancerproteasomal degradation ofthe BRCA1 tumorsuppressor is regulatedduring cell cycleprogression.SUMOylationCancer / tumorUbc9 (E2 conjugating35A role for Ubc9 inenzyme)tumorigenesisUbiquitinationAlzheimers diseaseBcl-236Inhibition of the ubiquitin-proteasome system inAlzheimer's DiseaseGlycosylationAlzheimers diseasetau37Glycosylation ofmicrotubule-associatedprotein tau: an abnormalposttranslationalmodification inAlzheimer's diseaseK48-linked and K63-linkedParkinson's diseasesynphilin-1, parkin, α-38Parkin mediated lysine 63-ubiquitinationsynuclein, UCHL1linked polyubiquitination: alink to protein inclusionsformation in Parkinson'sand other conformationaldiseases?UbiquitinationParkinson's Disease,Parkin39Parkin-mediated K63-Autophagylinked polyubiquitination: asignal for targetingmisfolded proteins to theaggresome-autophagypathway.UbiquitinationNeurodegenerativeP6240Lysine 63-linkedDiseasespolyubiquitin potentiallypartners with p62 topromote the clearance ofprotein inclusions byautophagy.Acetylation, deacetylation,Neurologic andHDAC41Epigenetic targets ofmethylationpsychiatric disordersHDAC inhibition inincluding Huntington'sneurodegenerative anddisease, Parkinson'spsychiatric disorders.disease, anxiety andmood disorders,Rubinstein-Taybisyndrome, and RettsyndromeNedd8ylationNeurodegenerativeNEDD842Accumulation of NEDD8Diseases, Parkinson'sin neuronal and glialdisease and Rosenthalinclusions offibres in astrocytomaneurodegenerativedisorders.NeurodegenerativeMad2, BubR143Inhibitory factorsdiseasesassociated to cDc20associated with anaphase-promotingcomplex / cylosome inmitotic checkpoint.UbiquitinationCell Cycle progression44Ubiquitin dependence ofselective proteindegradation demonstratedin the mammalian cellcycle mutant ts85.Cell Cycle progressioncyclin45Cyclin: a protein specifiedby maternal mRNA in seaurchin eggs that isdestroyed at each cleavagedivision.Cell Cycle progressionAPC / C (cDc20, CDH146Control of mitoticand MAD2)transitions by the anaphase-promoting complex.ConjugationCell Cycle progressioncyclin47Cyclin is degraded by theubiquitin pathway.UbiquitinationCell Cycle progressionCdc34, CDK activity-48How proteolysis drives theby degrading CDKcell cycleactivators or inhibitorsUbiquitinationCell Cycle progressionAPC / C (cDc20,49Ubiquitination by theMAD2)anaphase-promotingcomplex drives spindlecheckpoint inactivation.Ubiquitination, phosphorylation,DNA damage and repairATR / MRN complex50Twists and turns in themethylation DNA damage andfunction of DNA damagerepairsignaling and 5 repairproteins by PTMs.Acetylation, methylation,Huntington diseaseHistone (H2A, H2B,51Mechanisms of disease:phosphorylation, ubiquitinationH3 and H4)Histone modifications inand SUMOylationHuntington's disease.SUMOylationHuntington diseaseHuntingtin (Httex1p)52SUMO modification ofHuntingtin andHuntington's diseasepathology.Ubiquitination, SUMOylation,NFkB pathwayIkappaB kinase (IKK)53PTMs regulating thephosphorylation, acetylation andcomplex, the IkappaBactivity and function of thenitrosylationproteins and the NF-nuclear factor kappa BkappaBpathway.SUMOylationNeuronal IntranuclearSUMOylation54SUMOylation substrates inInclusion disease (NIID)substrates:neuronal intranuclearPromyelocyticinclusion disease.leukaemia protein(PML) andRanGAP1.HDAC4SUMOylationType 1 diabetesM55V substitution of55SUMO wrestling with typeSUMO41 diabetes.SUMOylationPolyglutamine DiseasesESCA1 and ESCA256Enhanced SUMOylation inpolyglutamine diseasesUbiquitinationKidney cancersHIF-alpha57The role of von Hippel-Lindau tumor suppressorprotein and hypoxia inrenal clear cell carcinoma.Neddylation, SUMOylation,Renal cell carcinomas,pVHL, NEDD858The von Hippel-Lindaupheochromocytomas,conjugation to Cul-2tumor suppressor geneand vascular tumors ofproduct promotes, but isthe central nervousnot essential for, NEDD8systemconjugation to cullin-5 2.SUMOylationDiabetes mellitus,ERK5, Ubc9 (SUMO59Effects of MEK5 / ERK5diabetic ventricularE2 conjugase) orassociation on smalldysfunctionPIAS1 (E3 ligase)ubiquitin-relatedmodification of ERK5:implications for diabeticventricular dysfunctionafter myocardial infarction.Ubiquitination, SUMOylationParkinson's,αSYN (PARK1), UCH-60The ubiquitin proteasomeAlzheimer's,L1, DJ-1 binds to thesystem inHuntington's, Prion andSUMO E3 PIASx, Aβneurodegenerativeamyotrophic lateraland tau, UBB + 1etc . . .diseases: sometimes thesclerosischicken, sometimes theegg.Methylation, deimination, andMultiple SclerosisMBP61Multiple sclerosis: anphosphorylationimportant role for PTMs ofmyelin basic protein inpathogenesis.GlycosylationAutoimmunity,IgG and IgA162Plasma proteinsRheumatoid arthritis andglycosylation and itsIgA nephropathyalteration in disease.SUMOylationParkinsonDJ-163Proper SUMO-1conjugation is essential toDJ-1 to exert its fullactivities.SUMOylationParkinsonDJ-1, and pyrimidine64DJ-1 transcriptionally up-tract-binding protein-regulates the humanassociated splicingtyrosine hydroxylase byfactor (PSF)inhibiting the sumoylationof pyrimidine tract-bindingprotein-associated splicingfactor.Ubiquitination, phosphorylationCancerp5365Ubiquitination,and acetylationphosphorylation andacetylation: the molecularbasis for p53 regulation.PhosphorylationCancerFra-166Accumulation of Fra-1 inras-transformed cellsdepends on bothtranscriptionalautoregulation and MEK-dependent posttranslationalstabilization.PhophorylationCancerNF-kappa B67Inhibition of constitutiveNF-kappa B activity by Ikappa B alpha Msuppresses tumorigenesis.Ubiquitination, SUMOylationCancerSmad468Sumoylation of Smad4, thecommon Smad mediator oftransforming growthfactor-beta familysignaling.PhophorylationUterine leiomyomasRef-169Altered PTM of redoxfactor 1protein in humanuterine smooth muscletumors.Phophorylationtumorigenesis,p53, GSK3beta70Glycogen synthase kinase3differentiation andbeta phosphorylates serineapoptosis33 of p53 and activatesp53's transcriptionalactivity.Phophorylationtumorigenesispp60c-src71pp60c-src in humanmelanocytes and melanoma30 cells exhibits elevatedspecific activity andreduced tyrosine 530phosphorylation comparedto human fibroblast pp60c-src.PhophorylationtumorigenesisP12072Abelson murine leukemiavirustransformationdefectivemutants with impairedP120 associated proteinkinase activity.GlycosylationPrion DiseasePrP73Asparagine-linkedglycosylation of the scrapieand cellular prion proteins.UbiquitinationFanconi anemiaFANCD274Fanconi anemia: causesand consequences ofgenetic instability.UbiquitinationFanconi anemiaFANCD2, catalytic75A novel ubiquitin ligase issubunit PHF9(FANCL)deficient in Fanconianemia.UbiquitinationAgingBRCA1; PCNA;76Aging and theNFκB; p27;ubiquitinome: traditionalSNEVPrp19 / Pso4and non-traditionalfunctions of ubiquitin inaging cells and tissues.Ubiquitination, SUMOylation,Aging77Aging and dietaryOxydationrestriction effects onubiquitination,sumoylation, and theproteasome in the heart.UbiquitinationAgingDAF-16, RLE-1 (E378RLE-1, an E3 ubiquitinligase)ligase, regulates C. elegansaging by catalyzing DAF-16 polyubiquitination.SUMOylationAgingPOMP-179Effects of aging and dietaryrestriction onubiquitination,sumoylation, and theproteasome in the spleen.AgingDecrease of expressed80Caretaker or undertaker?ProteasomeThe role of the proteasomeproteins: S9: Rpn6in aging(p44.5), Rpn5 (p55), a2(HC3), a7(HC8),S7: Rpt1 (MSS1) andS10b: Rpt4 (p42)S-nitrosylation, UbiquitinationParkinson's diseaseparkin81Nitrosative stress linked tosporadic Parkinson'sdisease: S-nitrosylation ofparkin regulates its E3ubiquitin ligase activity.GlycosylationVirus related diseasespenv9, penv1482Glycosylation inhibitorsblock the expression ofLAV / HTLV-III (HIV)glycoproteins.GlycosylationVirus related diseasesgp4683Immunogenicity andconformational propertiesof an N-linked glycosylatedpeptide epitope of humanT-lymphotropic virus type1 (HTLVI).GlycosylationVirus related diseasesperoxiredoxin 1 and84PosttranslationalHTLV-1-p24-(gag)glycosylation of targetproteins implicatemolecular mimicry in thepathogenesis of HTLV-1associated neurologicaldisease.GlycosylationVirus related diseasesgp 10085A glycopolypeptide (gp100) is the main antigendetected by HTLV-IIIantisera.Citrullination / deiminationMultiple Sclerosis,Myelin basic protein86A tale of two citrullines-Diabetes, Alzheimer's(MBP)structural and functionalaspects of myelin basicprotein deimination inhealth and 5 disease.OGlcNAcCardiac dysfunctionSP1, eNOS,87O-GlcNAc modification ofnucleocytoplasmic proteinsand diabetes.OGlcNAcDiabetes, Alzheimer'stau, β-amyloid88O-GlcNAc modification indiseaseprecurssor, AP-3,diabetes and Alzheimer'ssynapsin-I,disease.Neurofilament H, L, M.IRS, GS, PDX-1,eNOS, SP1OGlcNAcDiabetes89A bittersweet modification:O-GlcNAc and cardiacdysfunction.OGlcNAcDiabetesSp1(but also90PTM by O15 GlcNAc:metionned the serumanother way to changeresponse factor, c-myc,protein function.estrogen receptors andRNA pol II)Various PTMsAtherosclerosis; celiacαB-crystallin, MBP,91Posttranslational proteindisease; autoimmuneFibrin, Type IImodifications: new flavorsencephalomyelitis;collagen, MBP Ac1-1,in the menu ofmultiple sclerosis;Sm D1, D3, Wheatautoantigens.systemic lupusgliadin, LDL, SnRNPerythematosus; collagen-Dinduced arthritis;rheumatoid arthritisVarious PTMsMultiple sclerosis / EAE,Fillagrin, Vimentin,92PosttranslationalCollagen-inducedH2Bmodifications of self-arthritis, Rheumatoidantigens.arthritis, systemic lupuserythematosus.Various PTMsRheumatoid arthritis;trichohyalin, filaggrin93Modifications of argininesMultiple sclerosis;and keratin, myelinand their role inSystemic lupusbasic protein(MBP),autoimmunity.erythematosusfibrin, vimentin andnucleophosmin / B23,histones, Sm-D1, Sm-D3, Sm-ByB9, LSm4CitrullinationRheumatoid arthritisFibrin94Autoantigenicposttranslationalmodifications of proteins:does it apply to rheumatoidarthritis?

[0140] The methods of the invention can be applied to identify a set of biomarkers for a disease or medical condition. The set of biomarkers can include information such as the identity of two or more proteins whose level of a given PTM is altered (i.e., either increased, decreased, or modified in terms of the number or position of attached modifying moieties) in the disease or medical condition. The set can be established, for example, by comparing the protein PTM profile of one or more patients having the disease or medical condition with similar profiles from one or more control subjects who do not have the disease or medical condition. The profiles are obtained by separately contacting functional extracts from the patients and control subjects with a microarray containing an ordered plurality of proteins, such as proteins encoded by the human genome, and determining the level of PTM of one or more proteins in the microarray. The presence or absence, or the observed level, of PTM of proteins in the microarray for the patients is then compared with the presence or absence or level of PTM of the corresponding proteins for the control subjects. A set of biomarkers is formed from proteins of the patients whose level of PTM is altered compared to control levels. The biomarker set in some cases can be specific for a certain type of patient sample (e.g., plasma, cerebrospinal fluid, tissue, or cell type). Biomarker sets so identified can be used in any of the methods according to the invention, e.g., in a method of diagnosis.

[0141] Methods of the invention can be used to screen for and identify substrates of protein modifying enzymes. For example, a protein microarray containing a set of proteins that include candidate proteins for one or more selected types of PTM can be incubated with a solution containing one or more enzymes that catalyze PTM reactions. The methods described above can be employed to label and identify proteins in the array that serve as substrates for the enzyme(s). Optionally, the array can include variations of one or more protein substrates, e.g., sequence variants or proteins having one or more known modifications at different sites. The array can include only a single protein and its variants, or it can include proteins representative of an entire genome, or proteins expressed by a given cell or tissue, or any subset thereof. Such screening methods can be used to define the specificity of a protein modifying enzyme with respect to protein substrates or with respect to the enzyme recognition sequence, for example, or to analyze signaling pathways.

[0142] A further use for the methods of the invention is to characterize the activity of one or more protein modifying enzymes in a functional extract. A functional extract can be analyzed using methods described earlier, while supplementing only with chemical compounds that supply energy for the PTM reaction carried out by a particular enzyme or which serve as cofactors. The protein substrates for the enzyme are supplied in the protein microarray. Further characterization of the functional extract can then be obtained by supplementing it with one or more protein modifying enzymes. Depending on the nature of the signaling pathway, the functional extract can be supplemented with additional enzymes in different combinations in parallel assays. For example, in the case of polyubiquitination, one assay can be performed with the functional extract alone (i.e., no supplementation with exogenous enzymes), another assay can involve the supplementation of the functional extract with an E1 enzyme, and additional assays can involve supplementation with an E1 enzyme plus different combinations of E2 enzymes. In this way a full signaling pathway or any portion thereof can be characterized for a given functional extract using a large number of potential protein substrates by performing only a few reactions.

[0143] The invention also includes kits that are useful in practicing the methods presented here, e.g., diagnostic kits. A kit for the diagnosis of a disease or medical condition by the analysis of a PTM state of a protein in a patient sample contains a standard set of one or more functional extracts capable of producing a known pattern of protein PTM states on a protein microarray. Optionally, the kit also contains instructions for carrying out one or more of the methods outlined above. The kit can also optionally contain one or more reagents, such as substrates, co-factors, biochemical agents, buffers, enzymes, enzyme inhibitors, antibodies, or labeling moieties such as fluorophores or radiolabeled compounds. The kit also can include computer software for analysis, one or more protein microarrays, blocking reagents for such microarrays, and packaging material for any of the kit components.

[0144] Previous protein-based diagnostic tests typically have assayed the abundance of a protein, and in certain cases its activity. However, the present invention is unique in utilizing functional samples from patients to determine global PTMs or PTM alterations for diagnostics purposes. These methods may serve both for diagnosis of different diseases as described herein, and as a tool for the discovery of new biomarkers and drug targets.

[0145] There are many assays available to detect binding interactions, but up to now they have used either dilute protein solutions or detergent-containing cell lysates. The number and strength of the interactions detected are therefore distorted by the change in relative concentration of ligand and target, or by the presence of detergents. In addition, the modification profile can be affected by a change in the relative amounts of, for example, kinase / phosphatase pairs. In the methods according to the present invention, however, undiluted extract (functional extract) can be used without adding detergent, preserving the original physiological state. In addition to examining cytoplasmic fractions, nuclear fractions and smaller organelles can be applied to the microarray as well.

[0146] The present methods have far greater dynamic range than available mass spectrometry methods, since thousands of proteins can be spotted on an individual chip in pure form and at high concentration, removing the effect of their relative abundance. Proteins can also be attached to the microarray in different orientations to ensure that binding to different parts of the protein can be detected. The present methods are more straightforward compared to mass spectrometry, and considerably less time-consuming than SDS gels and similar techniques.

[0147] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0148] As used in this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “the method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0149] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.Example IProtein Ubiquitination Patterns Upon Escape from the Spindle Assembly Checkpoint in Mammalian Cells

[0150] Protein microarrays were used to identify the polyubiquitination state of proteins under specific cellular conditions. Highly concentrated cellular extracts that have demonstrable function specific for a particular phase of the cell cycle were used to modify the polyubiquitination state of human proteins on a microarray.

[0151] Specifically, the degradation of proteins involved in mitosis was examined by determining the polyubiquitination state of certain proteins at specific stages of the cell cycle. During mitosis, rapid degradation of the mitotic cyclins (11, 12) causes abrupt shut-down of mitotic kinase activity, allowing the cell to enter anaphase. The Anaphase Promoting Complex (APC), a multi-subunit E3 ligase, targets cyclins and other mitotic substrates for proteasomal degradation (13, 14) which in turn leads to the metaphase to anaphase transition. Thus, cell division is highly controlled by the degradation of polyubiquitinted proteins (15).

[0152] The experimental strategy was to use nocodazole arrested HeLa S3 functional cytoplasmic extracts and to follow protein polyubiquitination during release from the checkpoint by incubation on protein microarrays by assaying reactivity with labeled antibodies against polyubiquitin chains. Differentially modified proteins were examined in APC-inhibited versus APC-active extracts. The polyubiquitin signature of G1 extracts was also examined.Tissue Culture and Cell Synchronization

[0153] HeLa S3 cells were synchronized in prometaphase by treatment with nocodazole, or in G1 by a release from nocodazole arrest. Cells were incubated in thymidine-containing (2 mM) medium, and then released into fresh medium, followed by a nocodazole arrest (0.1 g / ml). For G1 cells, nocodazole-arrested cells were released into fresh medium for 4 h. Cells were harvested, washed with phosphate buffered saline (PBS), and processed for extraction as described below.Extract Preparation

[0154] HeLa S3 cells were synchronized with thymidine for 20 hours, released for 3 hours, and then arrested in mitosis by the addition of nocodazole for an additional 11 hours. Synchronized cells (CP-extracts) were then harvested, washed with PBS, lysed in Swelling Buffer (25 mM HEPES pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM dithiothreitol, 1 tablet of Complete protease inhibitors (Roche)), and homogenized by freeze-thawing and passage through a needle. G1-extracts were prepared in the same manner with an additional 4 hour release from nocodazole arrest. Extracts were cleared by subsequent centrifugation (5 min at 5,000 r.p.m. followed by 60 min at 14,000 r.p.m.). Extract (20 μl) was supplemented with Degradation Cocktail (1 μL) containing 1.5 mg / ml ubiquitin (Boston Biochem), 150 mM creatine phosphate, 20 mM ATP (pH 7.4), 2 mM EGTA (pH 7.7), 20 mM MgCl2).Incubation of Extracts with Microarrays

[0155] Human PROTO-ARRAY® microarrays (Invitrogen) were washed three times (10 min each) with TBS containing 0.05% Tween 20 (TBS-T) and then blocked for 4 hours at 4° C. with microarray blocking solution (ARRAYIT® brand BLOCKIT™ (TeleChem International, Inc.)). Extracts were pre-incubated with either Emi1 (1 mg / ml) or H20 for 30 minutes. 100 μl of CP or G1 extracts (~25 mg / nil) were then supplemented with UbcH10 (5 μl, 1 mg / ml; Boston Biochem) and incubated under a coverslip on the microarrays for 1 hour at RT. The arrays were then washed and incubated overnight with anti-polyubiquitin antibody (FK1, 1 mg / ml; Biomol) diluted 1:250. To label modified (polyubiquitinated) proteins, an anti-mouse Cy3-conjugated secondary antibody (3 μl; 1 mg / ml, Jackson ImmunoResearch Laboratories) was incubated for 1 hour at RT. The arrays were washed again, spin-dried (200 g, 5 min) and scanned with a GenePix 4000B scanner.Images and Data Processing

[0156] Results were recorded as TIFF files and images were quantified using Gene Pix Pro 5 feature extraction software (version 4000B). Scanning parameters were set so that none of the spots showed saturation: PMT gain value=400; laser power=30% (see FIG. 10). For each spot, the local background intensity was subtracted from the median spot intensity.Data Filtering and Normalization

[0157] The processed data set was organized in a matrix where each column contains the reactivities measured for a given array and each row contains the reactivities measured for a given protein over all arrays. The negative values were set to zero and the data was then normalized using the quantile normalization algorithm (32).Data Analysis

[0158] To determine subsets of proteins that were differentially modified on the different microarrays a two-sample t-test was used. Each protein was tested separately by comparing its signal intensity values in two different conditions (2 replicates per chip; 2 chips for each tested condition). Thus four signal intensities were measured for each protein and each condition. 1000 permutations were performed (within rows, i.e., all values for each protein were shuffled) and permutation-based p-values were calculated based on the new t-scores. P-values lower than 0.01 were considered significant.Degradation Assays

[0159] Coupled in vitro transcription and translation were performed from pCS2+ constructs using a rabbit reticulocyte lysate system (TnT SP6, Promega) or wheat germ extracts. 35S-labelled substrates were added to G1 or CP extracts of synchronized HeLa S3 cells (see extract preparation). Aliquots were removed at 0, 30, 60, and 90 min and analyzed by SDS-PAGE (4-15%) and autoradiography. Additionally, endogenous protein levels (actin (Sigma), securin (Mb1), calmodulin (Upstate), and p27 (Upstate)) were determined in the extracts by Western blotting at the indicated times.Results

[0160] The E2-conjugating enzyme, UbcH10, has been shown to overcome the metaphase-anaphase transition (16). After arresting cells in nocodazole, concentrated extracts (apprx 25 mg / ml) were made and these retain the checkpoint state (CP extracts). It is known that addition of UbcH10 to a concentration of 5 uM (approx. 25 mg protein / ml) to nocodazole-arrested, concentrated cell extracts inactivates the metaphase state and leads to APC-dependant substrate degradation (17).

[0161] Extracts were prepared from synchronized HeLa S3 cells arrested in mitosis or in G1. CP extracts were divided into three aliquots; one was retained, one was supplemented with UbcH10, and the third received UbcH10 and an inhibitor of APC, emi1. The samples were placed on the protein microarray for 60 minutes at room temperature (FIG. 2B). In order to control for the activity of the extracts, an aliquot of each sample was removed and 35S labeled-securin, a well-characterized APC substrate, was added to record its degradation (FIG. 2A). Securin remained stable in CP extracts even after 60 minutes at room temperature (FIG. 2A, right panel) which is consistent with the inhibition of APC by the spindle checkpoint. CP extract supplemented with UbcH10 (CP-released) degraded securin rapidly while the addition of the APC inhibitor Emi1 (APC-inhibited) stabilized securin for at least sixty minutes. To label modified proteins on the arrays, an anti-polyubiquitin antibody (FK1) was used (FIG. 5) with a Cy3-conjugated secondary antibody. Microarrays were then scanned and the median signal intensity and local background of each spot was measured. FIG. 2B illustrates the process and depicts one representative scanned subarray (out of 48 on each chip) and its reactivity.

[0162] Most of the spots in the microarray revealed a signal of low intensity or similar to the background level. Only 9-11% of the spots on each chip gave a positive signal after subtracting the local background intensity. FIG. 3A shows the distribution of the data of two representative chips under the CP-released (left panel) and APC-inhibited condition (right panel); the inset depicts the positive signal reactivity that was detected. A commonly accepted criterion for determining minimum signal (threshold) that can be accurately quantified is the measure of Signal to Noise Ratio (SNR) where a higher SNR indicates higher signal over background noise; a signal-to-noise ratio of 3 is commonly considered the lower limit for accurate detection. Thus, the SNR ratio for every spot on the chip was calculated as follows: SNR=(signal mean−background mean) / (standard deviation of the background) (18). Even though the background signal within each microarray was variable (FIG. 6), the SNR per spot revealed a clear signal (SNR>3) even for spots with a low signal intensity of about 1500 units (FIG. 7).

[0163] The threshold level defining a significant polyubiquitination signal was determined using the signal from 96 ‘buffer’ spots on each microarray. When subtracting the local background from the signal, 99% of the buffer spots on each chip gave a negative value (mean value of −1130; see FIG. 8). The signal of thirteen known APC substrates was determined on each chip was compared with the signal of the ‘buffer’ spots located adjacent to them (i.e., in the same subarray). As shown in FIG. 3B, nine of these substrates appeared to have a signal that was significantly higher than the buffer spots (p<0.05) but only five of them gave a positive signal. In order to reduce the potential false positive rate, only positive values were considered as reflecting real modification signals in this study.

[0164] To test the reproducibility of the assay and its ability to detect differential PTMs between different conditions, microarrays that were incubated with different extract preparations (biological replicates) were compared, and microarrays with extracts under different conditions (CP released vs. APC-inhibited) were also compared. FIG. 3C depicts the scatter plots of the positive spot reacitivities in each comparison (log scale). Visually the two different conditions (red dots) produced a signal that was more spread and variable compared to the biological replicates (black dots), which are closer to the diagonal. These distributions differ very significantly by statistical tests. Two microarrays were compared from each condition, and the p-value of the differences between corresponding proteins (each comprised of 4 spots) was calculated using a two-sample t-test. To control for the multiple hypothesis testing, the p-value determination was based on 1000 permutations (per protein) of the data. More than a hundred proteins yielded a significant p-value (p<0.01); these proteins are listed in Table 4. While these proteins varied greatly in their attributed functions and cellular processes, several known APC substrates are among the significantly detected proteins, including all three aurora kinases. Given the state of knowledge of APC substrates it was to be expected that some new substrates should have been detected by this approach. Five proteins (Nek9, Calm2, RPS6KA4, cyclin G2 and p2′7) that were detected as differentially modified in these microarrays had previously been reported to play a role in mitosis. These five proteins, together with two proteins (Zap-70 and MAP3K11) that were not previously shown to be involved in mitosis, were selected for a biochemical assay to test their ability to serve as APC substrates. Zap-70 and MAP3K11 showed no detectable ubiquitination or degradation in the biochemical assay for mitosis dependent degradation. It should be noted that not all substrates would be expected to score in such an assay, due to lack of cofactors, poor folding, lack of posttranslational modification, or other factors, and therefore a negative result is not dispositive. However, Nek9, Calm2, RPS6KA4 and cyclin G2 proteins were found to be degraded in the CP extracts, and their degradation was inhibited by the addition of emi1 (FIG. 4A). Interestingly, p27 appeared to be degraded in the CP-released extracts as well; however, a longer exposure (FIG. 4B) revealed that the protein accumulated polyubiquitin chains (causing a gel shift) and was not rapidly degraded (compare with the addition of the proteasome inhibitor MG-132). While the addition of emi1 did not inhibit completely the formation of ubiquitin chains, it appeared to yield a lower signal then seen in the CP-released extract; this conjugation might have occurred during the pre-incubation of the emi1 with the extracts. The endogenous level of calm2 and p27 in CP-released and APC-inhibited extracts was examined by Western blot. Both p27 and calm2 were degraded in the extracts from cells released into an anaphase-like state, and their degradation was inhibited by the addition of emi1.

[0165] TABLE 4Protein NameAccessionp-valuehistone UNFRAC. WHOLE HISTONE - known Autoantigen0.0002ring finger protein 128 (RNF128) transcript variant 1NM_194463.10.0004erythrocyte membrane protein band 4.1 like 5BC054508.10.0004BC013173 Homo sapiens, clone MGC: 17340BC013173.10.0004Clmodulin 2NM_0017430.0005HTGN29 protein (HTGN29)NM_020199.10.0006ankyrin repeat domain 13BC032833.20.0006ribosomal protein S6 kinase 90 kDa polypeptide 4 (RPS6KA4) transcript variant 2NM_001006944.10.0007macrophage stimulating 1 receptor (c-met-related tyrosine kinase) (MST1R)NM_002447.10.0008hypothetical protein FLJ11184BC011842.20.0008PCTAIRE protein kinase 2BC033005.10.0008aurora kinase A (AURKA) transcript variant 2NM_003600.20.0009dolichyl-phosphate mannosyltransferase polypeptide 2 regulatory subunit (DPM2)NM_152690.10.0009transcript variant 2ems1 sequence (mammary tumor and squamous cell carcinoma-associated (p80 / 85 srcNM_138565.10.0009substrate) (EMS1)cytochrome P450 family 26 subfamily A polypeptide 1 (CYP26A1) transcript variant 2NM_057157.10.0010KIAA0157 protein (KIAA0157)NM_032182.20.0010solute carrier family 23 (nucleobase transporters) member 2BC013112.20.0011ring finger protein 111BC060862.10.0011additional sex combs like 1 (Drosophila)BC064984.10.0012cDNA clone MGC: 39273 IMAGE: 5440834BC024289.10.0012PAS domain containing serine / threonine kinase (PASK)NM_015148.10.0013YY1 transcription factor (YY1)NM_003403.30.0013proteasome (prosome macropain) 26S subunit non-ATPase 4 (PSMD4) transcript variant 1NM_002810.10.0014hypothetical protein LOC143458 (LOC143458)NM_174902.20.0014selectin ligand interactor cytoplasmic-1 (SLIC1) transcript variant 1NM_153337.10.0015MAX interacting protein 1 (MXI1) transcript variant 2NM_130439.10.0015neural precursor cell expressed developmentally down-regulated 8 (NEDD8)NM_006156.10.0016aurora kinase B (AURKB)NM_004217.20.0016src homology three (SH3) and cysteine rich domainBC020221.10.0016hypothetical protein DKFZp762O076 (DKFZp762O076)NM_018710.10.0016Nedd4 family interacting protein 1 (NDFIP1)NM_030571.20.0016hypothetical protein FLJ36175BC029520.10.0017EGF-like repeats and discoidin I-like domains 3BC053656.10.0018hypothetical protein MGC4618 (MGC4618)NM_032326.10.0019zeta-chain (TCR) associated protein kinase 70 kDa (ZAP70) transcript variant 1NM_001079.30.0019ribosomal protein L30 (RPL30)NM_000989.20.0019feline sarcoma oncogene (FES)NM_002005.20.0019met proto-oncogene (hepatocyte growth factor receptor) (MET)NM_000245.20.0021ADP-ribosylation factor-like 7 (ARL7)NM_005737.30.0022Histone_F2a2 H2a(f2a2) - known Autoantigen0.0022likely ortholog of mouse gene trap locus 3 (GTL3)NM_013242.10.0022immediate early response 3 (IER3) transcript variant shortNM_003897.20.0023potassium voltage-gated channel shaker-related subfamily beta member 2 (KCNAB2)NM_003636.10.0023immunoglobulin heavy constant gamma 1 (G1m marker)BC014667.10.0024ring finger protein 4 (RNF4)NM_002938.20.0025proteasome (prosome macropain) 26S subunit non-ATPase 4 (PSMD4) transcript variant 2NM_153822.10.0026chromosome 6 open reading frame 145 (C6orf145)NM_183373.20.0027neurotrophic tyrosine kinase receptor type 1 (NTRK1) transcript variant 3NM_001007792.10.0028pleckstrin homology domain containing family G member 5 (PLEKHG5) transcriptNM_020631.20.0028variant 1Sjogren syndrome antigen A1 (52 kDa ribonucleoprotein autoantigen SS-A / Ro) (SSA1)NM_003141.20.0028interferon stimulated gene 20 kDa (ISG20)NM_002201.30.0028WD repeat domain 45 (WDR45) transcript variant 1NM_007075.30.0029TANK-binding kinase 1 (TBK1)NM_013254.20.0029chromosome 16 open reading frame 5BC002882.10.0030insulin-like growth factor 1 receptor (IGF1R)NM_000875.20.0030ring finger protein 111BC010369.10.0031G protein-coupled receptor kinase 4 (GRK4) transcript variant 2NM_001004056.10.0032v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN)NM_002350.10.0033RAS-like family 10 member BBC041133.10.0034hypothetical protein MGC11257 (MGC11257)NM_032350.30.0035chromosome 7 open reading frame 2 (C7orf2)NM_022458.20.0035expressed in T-cells and eosinophils in atopic dermatitis (ETEA)NM_014613.10.0036mitogen-activated protein kinase kinase kinase 11 (MAP3K11)NM_002419.20.0036casein kinase 1 alpha 1 (CSNK1A1) transcript variant 1NM_001025105.10.0038zeta-chain (TCR) associated protein kinase 70 kDa transcript variant 1BC053878.10.0038hypothetical gene LOC128439 (LOC128439)NM_139016.20.0038hypothetical protein MGC17403 (MGC17403)NM_152634.10.0039N-glycanase 1 (NGLY1)NM_018297.20.0039signal recognition particle 19 kDaBC010947.10.0040DNA fragmentation factor 40 kDa beta polypeptide (caspase-activated DNase) (DFFB)NM_001004285.10.0040transcript variant 3casein kinase 1 delta (CSNK1D) transcript variant 1 Not full-length.NM_001893.30.0042dendritic cell-derived ubiquitin-like protein (DC-UbP)NM_152277.10.0043cDNA clone MGC: 3432 IMAGE: 2959461BC013957.10.0043DnaJ (Hsp40) homolog subfamily B member 12 (DNAJB12) transcript variant 1NM_001002762.10.0043solute carrier family 36 (proton / amino acid symporter) member 4BC047374.10.0044SMT3 suppressor of mif two 3 homolog 1 (yeast) (SUMO1) transcript variant 1NM_003352.40.0044similar to hypothetical protein FLJ25555BC044239.10.0049lysosomal-associated protein transmembrane 4 alpha (LAPTM4A)NM_014713.20.0050KIAA1458 proteinBC031691.20.0051interleukin 17E (IL17E) transcript variant 1NM_022789.20.0053serum / glucocorticoid regulated kinase (SGK)NM_005627.10.0053hypothetical protein FLJ10156BC005004.10.0054thousand and one amino acid protein kinase (TAO1)NM_004783.10.0054ADP-ribosylation-like factor 6 interacting protein 4 (ARL6IP4)NM_016638.10.0054zinc finger protein 313 (ZNF313)NM_018683.20.0055solute carrier family 6 (neurotransmitter transporter) member 15BC022253.10.0055XM_378350.2XM_378350.20.0057low density lipoprotein receptor-related protein 10 (LRP10)NM_014045.10.0060arrestin domain containing 3 (ARRDC3)NM_020801.10.0062cyclin-dependent kinase inhibitor 1B (p27 Kip1) (CDKN1B)NM_004064.20.0062p53-regulated DDA3 (DDA3)NM_032636.20.0065calcium / calmodulin-dependent protein kinase IV (CAMK4)NM_001744.20.0066BC015569 Homo sapiens, Similar to SRp25 nuclear proteinBC015569.10.0066chromosome 6 open reading frame 201 (C6orf201)NM_206834.10.0067tripartite motif-containing 52 (TRIM52)NM_032765.10.0067hypothetical protein FLJ38628 (FLJ38628)NM_152267.20.0071vasopressin-induced transcriptBC000877.10.0074Ro-52 Ro-52 - known Autoantigen0.0074cyclin G2BC032518.10.0076mitogen-activated protein kinase kinase 6 (MAP2K6) transcript variant 2; mutantNM_031988.10.0077protein: MAP2K6 mutantconserved helix-loop-helix ubiquitous kinase (CHUK)NM_001278.30.0078aurora kinase C (AURKC) transcript variant 1NM_001015878.10.0079dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 (DYRK3) transcriptNM_001004023.10.0080variant 2cullin 3 (CUL3)NM_003590.20.0080hepatocyte growth factor-regulated tyrosine kinase substrate (HGS)NM_004712.30.0084B lymphoid tyrosine kinase (BLK)NM_001715.20.0084hypothetical protein MGC40579 (MGC40579)NM_152776.10.0086NIMA (never in mitosis gene a)-related kinase 9 (NEK9)NM_033116.20.0086solute carrier family 1 member 1 (SLC1A1) nuclear gene encoding mitochondrial proteinNM_004170.20.0086Homo sapiens, SWI / SNF related, matrix associated, actin dependent regulator ofBC018953.10.0086chromatin, subfamily d, member 2calcium binding protein 4BC033167.10.0088chromosome 19 open reading frame 28 (C19orf28)NM_174983.20.0088ubiquitin-activating enzyme E1-like (UBE1L)NM_003335.20.0090regenerating islet-derived 1 alpha (pancreatic stone protein pancreatic thread protein)BC005350.10.0090DnaJ (Hsp40) homolog subfamily B member 6 transcript variant 2BC000177.20.0091calcium / calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B)NM_001220.30.0093transcript variant1ubiquitin-conjugating enzyme E2-likeBC064566.10.0094melanoma antigen family B 1 (MAGEB1)NM_002363.10.0097secretory carrier membrane protein 3 (SCAMP3) transcript variant 1NM_005698.20.0097hypothetical protein LOC255330BC042038.10.0099 Example IIUbiquitination of Human Brain Proteins in Alzheimer's Disease

[0166] Human brain specimens are collected from deceased human subjects at autopsy after obtaining informed consent from the next of kin under protocols approved by the Partners Human Research Committee at Brigham and Women's Hospital. Weighed frozen human temporal or frontal cortices containing white and gray matter are added to freshly prepared, ice-cold TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) at a ratio of 4:1 (TBS volume / brain wet weight) and homogenized with 25 strokes at a setting of 10 on a mechanical Dounce homogenizer. The homogenate is centrifuged at 175,000×g in a TLA100.2 rotor on a Beckman TL 100 centrifuge, and then the supernatant is aliquoted and stored at −80° C.

[0167] For analysis of ubiquitination, samples are thawed on ice, supplemented with 5 μM ubiquitin, 2 mM ATP, and 150 mM creatine phosphate, and then incubated on a microarray to carry out the ubiquitination reaction. Optionally, E1 and / or E2 enzymes can be added to the extract, to determine if they are limiting the ubiquitination reaction.Example IIIProtein Ubiquitination in Cerebrospinal Fluid (CSF) from a Patient with Brain Tumor

[0168] Undiluted CSF from a patient with brain tumor was analyzed for enzyme activity responsible for PTM (ubiquitination) of human proteins. Conditions were similar to conditions used for cellular extracts. An ATP regenerating system and ubiquitin were added to the CSF sample, and the mixture was reacted with a protein microarray containing 8000 human proteins. A control reaction contained the same CSF sample but was not supplemented with ubiquitin or the energy mix.

[0169] A specific subset of proteins that are disproportionately expressed in brain (compared to a background of all the proteins that were on the chip) were found to be ubiquitinated (i.e., showed at least 2.5-fold higher signal than in the control), as shown in FIG. 11. The proteins that underwent CSF-mediated ubiquitination were distinct from background modification seen under control conditions. The functional annotation categories (gene ontology (‘GO’) terms) of these proteins were analyzed using the FatiGO online tool. List #1 shown in FIG. 11 holds the accession numbers for proteins that were highly ubiquitinated in comparison to the control (i.e. predicted list). List #2 holds the accession numbers of all the proteins on the microarray (i.e. background list). The ‘GO’ terms that are labeled with an asterisk (*) were shown to be enriched in this analysis, and the percentages of their appearance in the predicted list and in the background list is given in the third column. For comparison, terms associated with stress response (second row) showed no difference percentage of appearance in the ubiquitinated list when compared to the background list.Example IVAnalysis of Protein Ubiquitination in Normal Human Cerebrospinal Fluid (CSF) Sample

[0170] The ubiquitinating activity in a normal human CSF sample was tested by Western blotting. The ubiquitination reaction was started by adding an ATP regenerating system (2 mM ATP and 150 mM creatine phosphate) and ubiquitin (5 μM) to an aliquot of the CSF sample, and the reaction was run for 1 hour at 30° C. After the reaction was completed, the reaction mixture was subjected to SDS-PAGE and detection was performed with an anti-polyubiquitin antibody (FK1, Biomol). The results are shown in FIG. 12. There was a high molecular weight smear of ubiquitinated proteins in the reaction that included CSF and added ubiquitin, as compared to negative controls (CSF treated at 95° C. for 5 min or ubiquitin omitted).

[0171] Next, the ubiquitinating activity of CSF was tested by allowing it to ubiqutinate proteins in a human protein microarray. The CSF sample was supplemented with 2 mM ATP and 150 mM creatine phosphate and ubiquitin (5 μM). The sample was then incubated on a Human PROTO-ARRAY® (Invitrogen) protein microarray in order to identify the basal ubiquitination activity in the sample. After incubation of the samples on the arrays for 60 min at 25° C., the activity was stopped by washing the microarrays with TBS containing 0.05% Tween-20, and the modified proteins were identified using a first antibody specific for the polyubiqutinated state, and a second antibody (DyLight 649-conjugated goat anti-mouse IgM with minimal cross-reactivity to human, (catalog #115-495-075), Jackson ImmunoResearch) directed to the first antibody. The second antibody carried a fluorescent label (DyLight 649) for detection. The signal intensity of each spot in the microarray (reflecting the ubiquitination of the protein on that spot) was used to statistically identify ubiquitinated proteins (i.e., those spots having signal statistically significant over background fluorescence or a control spot). Ubiquitinated proteins in the array showed a difference of between 2- and 50-fold compared to a control reaction without added CSF (FIG. 13). The number of proteins that met the criteria ranged from 12 to 485 proteins in one CSF sample (lower line, •) and from 10 to 265 in the other (upper line, +). FIG. 14 presents a list of proteins that showed increased modification signal in each of the two CSF samples at a level of more than 50-fold when compared to the control (non-CSF) reaction, together with the fluorescence intensity of four spots for each protein. The scale indicates the value (log transformed) of each of the 4 duplicate spots of these proteins (2 microarrays; 2 spots per microarray, lanes 1-4) compared to the values on the control array on the right (lanes 5-6). A colorbar is given on the right (blue (bottom of the scale), low reactivity; red (top of the scale), high reactivity). A list of proteins that showed at least a 50-fold increase in their level of ubiquitination by the CSF (vs. no CSF) is presented in Table 5.

[0172] TABLE 5AccessionProtein DescriptionNM_006259S100 calcium binding protein A14 (S100A14), mRNANM_020672Williams Beuren syndrome chromosome region 22, mRNA(cDNA clone MGC: 2022 IMAGE: 3544156)BC001780zinc finger CCCH-type containing 10 (ZC3H10), mRNANM_032786chemokine (C-X-C motif) ligand 11 (CXCL11), mRNANM_032357ankyrin repeat and BTB (POZ) domain containing 1(ABTB1), transcript variant 1, mRNANM_006597interleukin 1, alpha (IL1A), mRNANM_032548v-akt murine thymoma viral oncogene homolog 3 (proteinkinase B, gamma) (AKT3), transcript variant 1, mRNANM_174902serine carboxypeptidase 1, mRNA (cDNA clone IMAGE:4328599), partial cdsNM_000961v-raf murine sarcoma 3611 viral oncogene homolog(ARAF), mRNANM_002609tec protein tyrosine kinase (TEC), mRNANM_025160myotilin (MYOT), transcript variant 1, mRNANM_017881platelet-derived growth factor receptor, beta polypeptide(PDGFRB), mRNANM_033505SELI selenoprotein I (SLE1)Example VProteins Modified with Ubiquitin-Like Modifiers Upon Mitotic Release

[0173] The PTM of human proteins in a microarray was studied using functional cell extracts from HeLa S3 cells obtained after release from the mitotic checkpoint (CP). Growth, cell cycle modulation, preparation of extracts of the cells, and microarray measurements were as described in Example 1. Separate reactions were performed using each of the following modifying moieties (ubiquitin-like modifiers): ubiquitin, sumo1, sumo2 / 3, FAT10, UFM1, and ISG15. Table 1 describes further details of selected ubiquitin-like modifiers. In each case, the cell extract was supplemented with energy mix plus 5 μM of the respective modifying moiety.

[0174] Checkpoint extracts from HeLa S3 cells arrested with nocodazole were divided into two aliquots, one was denoted as the checkpoint-arrested extract (CP-arrested), and one was supplemented with UbcH10 to relieve the checkpoint arrest (CP-released). Microarrays were incubated with these extracts to allow the proteins on the array to be modified. Each microarray contained approximately 8000 proteins spotted in duplicates at a reported level of around 10 pg per spot (median diameter approximately 150 μm). After washing the reaction off the microarray, an antibody specific to the modifying moiety used in the reaction was added to detect modified proteins on the microarray. Microarrays were scanned, and the median signal intensity and local background of each spot was measured. Then, the anti-modifier antibody was detected by adding a fluorescently-labeled secondary antibody. Microarrays were scanned and the median signal intensity and local background of each spot was measured. The data were then organized in a matrix where each column contains the reactivity measured for a given array, and each row contains the reactivity measured for a given protein over all arrays. The negative values were set to zero, and the data were then normalized using a quantile normalization algorithm. Table 6 summarizes the proteins that were either differentially modified in anaphase over metaphase or were highly modified. The highly modified (but not differentially modified) proteins are indicated with an asterisk, and the remaining proteins were differentially modified.

[0175] TABLE 6GenBankAccessionGene SymbolNameUbiquitinBC001396C9ORF32CHROMOSOME 9 OPEN READING FRAME 32BC004967UBAC1UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1BC007581ALDH4A1ALDEHYDE DEHYDROGENASE 4 FAMILY, MEMBERA1BC008720CRELD1DKFZP566D213 PROTEINBC010369RNF111RING FINGER PROTEIN 111BC011399SYKSPLEEN TYROSINE KINASEBC013173RSPRY1RING FINGER AND SPRY DOMAIN CONTAINING 1BC015219RBCK1CHROMOSOME 20 OPEN READING FRAME 18BC020221STACSH3 AND CYSTEINE RICH DOMAINBC021988NDFIP2NEDD4 FAMILY INTERACTING PROTEIN 2BC032518CCNG2CYCLIN G2BC036540LOC400120HYPOTHETICAL LOC400120BC041133RASL10BRAS-LIKE, FAMILY 10, MEMBER BBC044239ANKRD13DANKYRIN REPEAT DOMAIN 13 FAMILY, MEMBER DBC046151TOM1TARGET OF MYB1 (CHICKEN)BC048970TTLL7TUBULIN TYROSINE LIGASE-LIKE FAMILY,MEMBER 7BC056240SPRR1BSMALL PROLINE-RICH PROTEIN 1B (CORNIFIN)BC066340BLOC1S1BIOGENESIS OF LYSOSOME-RELATED ORGANELLESCOMPLEX-1, SUBUNIT 1NM_000875IGF1RINSULIN-LIKE GROWTH FACTOR 1 RECEPTORNM_001004056GRK4G PROTEIN-COUPLED RECEPTOR KINASE 4NM_001220CAMK2BCALCIUM / CALMODULIN-DEPENDENT PROTEINKINASE (CAM KINASE) II BETANM_002103GYS1GLYCOGEN SYNTHASE 1 (MUSCLE)NM_002378MATKMEGAKARYOCYTE-ASSOCIATED TYROSINEKINASENM_002648PIM1PIM-1 ONCOGENENM_002810PSMD4PROTEASOME (PROSOME, MACROPAIN) 26SSUBUNIT, NON-ATPASE, 4NM_003045SLC7A1SOLUTE CARRIER FAMILY 7 (CATIONIC AMINOACID TRANSPORTER, Y+ SYSTEM), MEMBER 1NM_003403YY1YY1 TRANSCRIPTION FACTORNM_004438EPHA4EPH RECEPTOR A4NM_004712HGSHEPATOCYTE GROWTH FACTOR-REGULATEDTYROSINE KINASE SUBSTRATENM_004783TAOK2TAO KINASE 2NM_005030PLK1POLO-LIKE KINASE 1 (DROSOPHILA)NM_005727TSPAN1TETRASPANIN 1NM_005737ARL4CADP-RIBOSYLATION FACTOR-LIKE 4CNM_006007ZFAND5ZINC FINGER, A20 DOMAIN CONTAINING 2NM_006293TYRO3TYRO3 PROTEIN TYROSINE KINASENM_013242C16ORF80GENE TRAP LOCUS 3 (MOUSE)NM_018215FLJ10781HYPOTHETICAL PROTEIN FLJ10781NM_018384GIMAP5GTPASE, IMAP FAMILY MEMBER 5NM_022905TTC23TETRATRICOPEPTIDE REPEAT DOMAIN 23NM_032182KIAA0157KIAA0157NM_032765TRIM52TRIPARTITE MOTIF-CONTAINING 52NM_080823SRMSSRC-RELATED KINASE LACKING C-TERMINALREGULATORY TYROSINE AND N-TERMINALMYRISTYLATION SITESNM_130439MXI1MAX INTERACTOR 1NM_152285ARRDC1ARRESTIN DOMAIN CONTAINING 1NM_153217TMEM174HYPOTHETICAL PROTEIN MGC13034NM_153822PSMD4PROTEASOME (PROSOME, MACROPAIN) 26SSUBUNIT, NON-ATPASE, 4NM_173541C10ORF91CHROMOSOME 10 OPEN READING FRAME 91NM_194271RNF34RING FINGER PROTEIN 34BC016381NANABC004967*UBAC1UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1BC010369*RNF111RING FINGER PROTEIN 111BC014475*BIRC7LIVIN INHIBITOR-OF-APOTOSISBC015569*ARL6IP4ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTINGPROTEIN 4BC021988*NDFIP2NEDD4 FAMILY INTERACTING PROTEIN 2BC023982*C5ORF32PUTATIVE NUCLEAR PROTEIN ORF1-FL49BC025700*AFF4AF4 / FMR2 FAMILY, MEMBER 4BC044239*ANKRD13DANKYRIN REPEAT DOMAIN 13 FAMILY, MEMBER DBC053895*IRS1INSULIN RECEPTOR SUBSTRATE 1BC054049*ZNF364ZINC FINGER PROTEIN 364BC060833*PRRG1PROLINE RICH GLA (G-CARBOXYGLUTAMIC ACID) 1NM_001033551*TOM1L2TARGET OF MYB1-LIKE 2 (CHICKEN)NM_002019*FLT1FMS-RELATED TYROSINE KINASE 1 (VASCULARENDOTHELIAL GROWTH FACTOR / VASCULARPERMEABILITY FACTOR RECEPTOR)NM_002110*HCKHEMOPOIETIC CELL KINASENM_002253*KDRKINASE INSERT DOMAIN RECEPTOR (A TYPE IIIRECEPTOR TYROSINE KINASE)NM_002938*RNF4RING FINGER PROTEIN 4NM_002944*ROS1V-ROS UR2 SARCOMA VIRUS ONCOGENE HOMOLOG1 (AVIAN)NM_002946*RPA2REPLICATION PROTEIN A2, 32 KDANM_005053*RAD23ARAD23 HOMOLOG A (S. CEREVISIAE)NM_005228*EGFREPIDERMAL GROWTH FACTOR RECEPTOR(ERYTHROBLASTIC LEUKEMIA VIRAL (V-ERB-B)ONCOGENE HOMOLOG, AVIAN)NM_012478*WBP2WW DOMAIN BINDING PROTEIN 2NM_017949*CUEDC1CUE DOMAIN CONTAINING 1NM_020182*TMEPAITRANSMEMBRANE, PROSTATE ANDROGENINDUCED RNANM_020630*RETRET PROTO-ONCOGENE (MULTIPLE ENDOCRINENEOPLASIA AND MEDULLARY THYROIDCARCINOMA 1, HIRSCHSPRUNG DISEASE)NM_030636*EEPD1KIAA1706 PROTEINNM_130465*TSPAN17TETRASPANIN 17NM_152267*RNF185RING FINGER PROTEIN 185NM_153229*TMEM92TRANSMEMBRANE PROTEIN 92NM_153345*TMEM139HYPOTHETICAL PROTEIN FLJ90586NM_194271*RNF34RING FINGER PROTEIN 34Sumo2 / 3NM_014805EPM2AIP1EPM2A (LAFORIN) INTERACTING PROTEIN 1NM_177974CASC4CANCER SUSCEPTIBILITY CANDIDATE 4BC017789CHORDC1CYSTEINE AND HISTIDINE-RICH DOMAIN (CHORD)-CONTAINING 1NM_018393TCP11L1T-COMPLEX 11 (MOUSE) LIKE 1NM_017588WDR5WD REPEAT DOMAIN 5BC056402LOC144097HYPOTHETICAL PROTEIN BC007540NM_003697OR5F1OLFACTORY RECEPTOR, FAMILY 5, SUBFAMILY F,MEMBER 1NM_014868RNF10RING FINGER PROTEIN 10NM_016269LEF1LYMPHOID ENHANCER-BINDING FACTOR 1BC014475BIRC7LIVIN INHIBITOR-OF-APOTOSISBC009207HIC2HYPERMETHYLATED IN CANCER 2NM_031845MAP2MICROTUBULE-ASSOCIATED PROTEIN 2BC020523INTS7CHROMOSOME 1 OPEN READING FRAME 73NM_018679TCP11T-COMPLEX 11 (MOUSE)NM_019087ARL15ADP-RIBOSYLATION FACTOR-LIKE 15BC043247TLE3TRANSDUCIN-LIKE ENHANCER OF SPLIT 3 (E(SP1)HOMOLOG, DROSOPHILA)BC002677AHDC1AT HOOK, DNA BINDING MOTIF, CONTAINING 1NM_003403YY1YY1 TRANSCRIPTION FACTORBC039583MGEA5MENINGIOMA EXPRESSED ANTIGEN 5(HYALURONIDASE)NM_015148PASKPAS DOMAIN CONTAINING SERINE / THREONINEKINASEBC010125C3ORF37CHROMOSOME 3 OPEN READING FRAME 37NM_001786CDC2CELL DIVISION CYCLE 2, G1 TO S AND G2 TO MBC005008CEACAM6CARCINOEMBRYONIC ANTIGEN-RELATED CELLADHESION MOLECULE 6 (NON-SPECIFIC CROSSREACTING ANTIGEN)NM_144706C2ORF15CHROMOSOME 2 OPEN READING FRAME 15NM_007277EXOC3EXOCYST COMPLEX COMPONENT 3NM_002648PIM1PIM-1 ONCOGENENM_002019FLT1FMS-RELATED TYROSINE KINASE 1 (VASCULARENDOTHELIAL GROWTH FACTOR / VASCULARPERMEABILITY FACTOR RECEPTOR)NM_152619DCLK2DOUBLECORTIN AND CAM KINASE-LIKE 2BC022253SLC6A15SOLUTE CARRIER FAMILY 6, MEMBER 15NM_017949CUEDC1CUE DOMAIN CONTAINING 1NM_006002UCHL3UBIQUITIN CARBOXYL-TERMINAL ESTERASE L3(UBIQUITIN THIOLESTERASE)NM_001278CHUKCONSERVED HELIX-LOOP-HELIX UBIQUITOUSKINASENM_001219CALUCALUMENINBC050645BYSLBYSTIN-LIKEBC040272IL16INTERLEUKIN 16 (LYMPHOCYTECHEMOATTRACTANT FACTOR)BC023152GYG2GLYCOGENIN 2NM_002011FGFR4FIBROBLAST GROWTH FACTOR RECEPTOR 4BC024725ANKRD50ANKYRIN REPEAT DOMAIN 50NM_138353LOC90379HYPOTHETICAL PROTEIN BC002926BC061697C3ORF62CHROMOSOME 3 OPEN READING FRAME 62NM_015417SPEF1CHROMOSOME 20 OPEN READING FRAME 28NM_181707C17ORF64CHROMOSOME 17 OPEN READING FRAME 64NM_199334THRATHYROID HORMONE RECEPTOR, ALPHA(ERYTHROBLASTIC LEUKEMIA VIRAL (V-ERB-A)ONCOGENE HOMOLOG, AVIAN)BC060760GIMAP6IMMUNE ASSOCIATED NUCLEOTIDE 2NM_002738PRKCB1PROTEIN KINASE C, BETA 1BC000247THAP4THAP DOMAIN CONTAINING 4BC013567USP48HYPOTHETICAL PROTEIN FLJ11328NM_198498C11ORF53CHROMOSOME 11 OPEN READING FRAME 53BC012289KIAA0515KIAA0515 PROTEINBC004219AGPAT31-ACYLGLYCEROL-3-PHOSPHATE O-ACYLTRANSFERASE 3NM_130766SKIPSKELETAL MUSCLE AND KIDNEY ENRICHEDINOSITOL PHOSPHATASENM_001328CTBP1C-TERMINAL BINDING PROTEIN 1BC058861SULT1C4SULFOTRANSFERASE FAMILY, CYTOSOLIC, 1C,MEMBER 2BC046117DNALI1DYNEIN, AXONEMAL, LIGHT INTERMEDIATEPOLYPEPTIDE 1NM_032017STK40SERINE / THREONINE KINASE 40NM_173822FAM126BHYPOTHETICAL PROTEIN MGC39518BC032120C20ORF11CHROMOSOME 20 OPEN READING FRAME 11NM_001556IKBKBINHIBITOR OF KAPPA LIGHT POLYPEPTIDE GENEENHANCER IN B-CELLS, KINASE BETANM_032014MRPS24MITOCHONDRIAL RIBOSOMAL PROTEIN S24NM_145796POGZPOGO TRANSPOSABLE ELEMENT WITH ZNFDOMAINNM_001042599ERBB4NM_017629EIF2C4ARGONAUTE 4NM_032846RAB2BRAB2B, MEMBER RAS ONCOGENE FAMILYBC011234SMNDC1SURVIVAL MOTOR NEURON DOMAIN CONTAINING 1NM_017583TRIM44TRIPARTITE MOTIF-CONTAINING 44NM_005639SYT1SYNAPTOTAGMIN INM_016954TBX22T-BOX 22NM_002796PSMB4PROTEASOME (PROSOME, MACROPAIN) SUBUNIT,BETA TYPE, 4NM_000666ACY1AMINOACYLASE 1NM_032326TMEM175HYPOTHETICAL PROTEIN MGC4618NM_001197BIKBCL2-INTERACTING KILLER (APOPTOSIS-INDUCING)NM_170672RASGRP3RAS GUANYL RELEASING PROTEIN 3 (CALCIUMAND DAG-REGULATED)BC017357ZNF765HYPOTHETICAL PROTEIN BC001610BC020233IGLC2IMMUNOGLOBULIN LAMBDA CONSTANT 1 (MCGMARKER)BC059374STK31SERINE / THREONINE KINASE 31NM_014248RBX1RING-BOX 1NM_005158ABL2V-ABL ABELSON MURINE LEUKEMIA VIRALONCOGENE HOMOLOG 2 (ARG, ABELSON-RELATEDGENE)NM_018668VPS33BVACUOLAR PROTEIN SORTING 33B (YEAST)BC063451TCP10L2T-COMPLEX 10 (MOUSE)NM_002623PFDN4PREFOLDIN SUBUNIT 4BC016652BMXBMX NON-RECEPTOR TYROSINE KINASENM_153486LDHDLACTATE DEHYDROGENASE DNM_033307CASP4CASPASE 4, APOPTOSIS-RELATED CYSTEINEPEPTIDASENM_004113FGF12FIBROBLAST GROWTH FACTOR 12NM_005148UNC119UNC-119 HOMOLOG (C. ELEGANS)NM_004838HOMER3HOMER HOMOLOG 3 (DROSOPHILA)NM_016355DDX47DEAD (ASP-GLU-ALA-ASP) (SEQ ID NO: 2) BOXPOLYPEPTIDE 47NM_014548TMOD2TROPOMODULIN 2 (NEURONAL)BC016964MRGPRFMAS-RELATED GPR, MEMBER FBC029220SOX5SRY (SEX DETERMINING REGION Y)-BOX 5BC030711C2ORF13CHROMOSOME 2 OPEN READING FRAME 13NM_001571IRF3INTERFERON REGULATORY FACTOR 3BC031830KLHL32KIAA1900NM_153498CAMK1DCALCIUM / CALMODULIN-DEPENDENT PROTEINKINASE IDNM_144602C16ORF78HYPOTHETICAL PROTEIN MGC32905NM_012325MAPRE1MICROTUBULE-ASSOCIATED PROTEIN, RP / EBFAMILY, MEMBER 1BC057840PSMB5PROTEASOME (PROSOME, MACROPAIN) SUBUNIT,BETA TYPE, 5NM_079422MYL1MYOSIN, LIGHT POLYPEPTIDE 1, ALKALI;SKELETAL, FASTBC029267MUC20MUCIN 20NM_020830WDFY1WD REPEAT AND FYVE DOMAIN CONTAINING 1NM_033003GTF2IBC009571STRA13STIMULATED BY RETINOIC ACID 13 HOMOLOG(MOUSE)NM_005030PLK1POLO-LIKE KINASE 1 (DROSOPHILA)NM_022754SFXN1LIKELY ORTHOLOG OF MOUSE SIDEROFLEXIN 1BC012997SULF1SULFATASE 1NM_001221CAMK2DCALCIUM / CALMODULIN-DEPENDENT PROTEINKINASE (CAM KINASE) II DELTABC031691SLAIN2KIAA1458 PROTEINNM_014840NUAK1NUAK FAMILY, SNF1-LIKE KINASE, 1BC001772QARSGLUTAMINYL-TRNA SYNTHETASENM_032693ARD1BBC025314IGHG1IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 1(G1M MARKER)BC033491ADAD2TESTIS NUCLEAR RNA-BINDING PROTEIN-LIKEBC009650PDS5ASCC-112 PROTEINNM_018326GIMAP4GTPASE, IMAP FAMILY MEMBER 4NM_005239ETS2V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENEHOMOLOG 2 (AVIAN)NM_006257PRKCQPROTEIN KINASE C, THETANM_152667NANPN-ACETYLNEURAMINIC ACID PHOSPHATASEBC001728*TFPTTCF3 (E2A) FUSION PARTNER (IN CHILDHOODLEUKEMIA)BC001772*QARSGLUTAMINYL-TRNA SYNTHETASEBC007048*ZMYM5ZINC FINGER, MYM-TYPE 5BC010125*C3ORF37CHROMOSOME 3 OPEN READING FRAME 37BC017314*ETS1V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENEHOMOLOG 1 (AVIAN)BC020985*COASYCOENZYME A SYNTHASEBC036572*ZCCHC12ZINC FINGER, CCHC DOMAIN CONTAINING 12BC040949*MEF2DMADS BOX TRANSCRIPTION ENHANCER FACTOR 2,POLYPEPTIDE D (MYOCYTE ENHANCER FACTOR2D)BC056402*LOC144097HYPOTHETICAL PROTEIN BC007540BC056415*RPAP3HYPOTHETICAL PROTEIN FLJ21908NM_001014796*DDR2DISCOIDIN DOMAIN RECEPTOR FAMILY, MEMBER 2NM_001039468*MARK2MAP / MICROTUBULE AFFINITY-REGULATINGKINASE 2NM_001786*CDC2CELL DIVISION CYCLE 2, G1 TO S AND G2 TO MNM_001910*CTSECATHEPSIN ENM_002378*MATKMEGAKARYOCYTE-ASSOCIATED TYROSINEKINASENM_002497*NEK2NIMA (NEVER IN MITOSIS GENE A)-RELATEDKINASE 2NM_002938*RNF4RING FINGER PROTEIN 4NM_003141*TRIM21TRIPARTITE MOTIF-CONTAINING 21NM_006257*PRKCQPROTEIN KINASE C, THETANM_006259*PRKG2PROTEIN KINASE, CGMP-DEPENDENT, TYPE IINM_006937*SUMO2SMT3 SUPPRESSOR OF MIF TWO 3 HOMOLOG 2(YEAST)NM_015981*CAMK2ACALCIUM / CALMODULIN-DEPENDENT PROTEINKINASE (CAM KINASE) II ALPHANM_016058*TPRKBTP53RK BINDING PROTEINNM_017838*NOLA2NUCLEOLAR PROTEIN FAMILY A, MEMBER 2(H / ACA SMALL NUCLEOLAR RNPS)NM_021709*SIVA1CD27-BINDING (SIVA) PROTEINNM_032752*ZNF496ZINC FINGER PROTEIN 496NM_130807*MOBKL2AMOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE2A (YEAST)NM_145173*DIRAS1DIRAS FAMILY, GTP-BINDING RAS-LIKE 1NM_175907*ZADH2HYPOTHETICAL PROTEIN BC010734NM_033003*NANANedd8BC000178KCMF1POTASSIUM CHANNEL MODULATORY FACTOR 1BC000395LETMD1LETM1 DOMAIN CONTAINING 1BC001852THG1LINTERPHASE CYCTOPLASMIC FOCI PROTEIN 45BC002526HSPA4HEAT SHOCK 70 KDA PROTEIN 4BC007312KIRREL2KIN OF IRRE LIKE 2 (DROSOPHILA)BC009074C8ORF70CHROMOSOME 8 OPEN READING FRAME 70BC009485C4ORF16CHROMOSOME 4 OPEN READING FRAME 16BC012945C19ORF57HYPOTHETICAL PROTEIN MGC11271BC018953SMARCD2SWI / SNF RELATED, MATRIX ASSOCIATED, ACTINDEPENDENT REGULATOR OF CHROMATIN,SUBFAMILY D, MEMBER 2BC020658TMEM40TRANSMEMBRANE PROTEIN 40BC038504SNF1LKSNF1-LIKE KINASEBC050696C12ORF48CHROMOSOME 12 OPEN READING FRAME 48BC051849RPAINRPA INTERACTING PROTEINBC062736CTD-2090I13.4BASIC TRANSCRIPTION FACTOR 3, PSEUDOGENE 9NM_004235KLF4KRUPPEL-LIKE FACTOR 4 (GUT)NM_004391CYP8B1CYTOCHROME P450, FAMILY 8, SUBFAMILY B,POLYPEPTIDE 1NM_005206CRKV-CRK SARCOMA VIRUS CT10 ONCOGENEHOMOLOG (AVIAN)NM_005651TDO2TRYPTOPHAN 2,3-DIOXYGENASENM_006251PRKAA1PROTEIN KINASE, AMP-ACTIVATED, ALPHA 1CATALYTIC SUBUNITNM_012328DNAJB9DNAJ (H5P40) HOMOLOG, SUBFAMILY B, MEMBER 9NM_013442STOML2STOMATIN (EPB72)-LIKE 2NM_014878KIAA0020KIAA0020NM_018014BCL11AB-CELL CLL / LYMPHOMA 11A (ZINC FINGERPROTEIN)NM_019895CLNS1ACHLORIDE CHANNEL, NUCLEOTIDE-SENSITIVE, 1ANM_021803IL21INTERLEUKIN 21NM_152443RDH12RETINOL DEHYDROGENASE 12 (ALL-TRANS AND 9-CIS)BC051366NANABC005008*CEACAM6CARCINOEMBRYONIC ANTIGEN-RELATED CELLADHESION MOLECULE 6 (NON-SPECIFIC CROSSREACTING ANTIGEN)BC006323*ABCB7ATP-BINDING CASSETTE, SUB-FAMILY B(MDR / TAP), MEMBER 7BC011707*NRBF2NUCLEAR RECEPTOR BINDING FACTOR 2BC012109*HOMER2HOMER HOMOLOG 2 (DROSOPHILA)BC020985*COASYCOENZYME A SYNTHASEBC021906*FMNL1FORMIN-LIKE 1BC053895*IRS1INSULIN RECEPTOR SUBSTRATE 1BC056669*DCUN1D2DCN1, DEFECTIVE IN CULLIN NEDDYLATION 1,DOMAIN CONTAINING 2 (S. CEREVISIAE)BC058924*UBE2MUBIQUITIN-CONJUGATING ENZYME E2M (UBC12HOMOLOG, YEAST)NM_001004105*GRK6G PROTEIN-COUPLED RECEPTOR KINASE 6NM_001039468*MARK2MAP / MICROTUBULE AFFINITY-REGULATINGKINASE 2NM_001798*CDK2CYCLIN-DEPENDENT KINASE 2NM_001895*CSNK2A1CASEIN KINASE 2, ALPHA 1 POLYPEPTIDENM_003141*TRIM21TRIPARTITE MOTIF-CONTAINING 21NM_003668*MAPKAPK5MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATED PROTEIN KINASE 5NM_005019*PDE1APHOSPHODIESTERASE 1A, CALMODULIN-DEPENDENTNM_005038*PPIDPEPTIDYLPROLYL ISOMERASE D (CYCLOPHILIN D)NM_006156*NEDD8NEURAL PRECURSOR CELL EXPRESSED,DEVELOPMENTALLY DOWN-REGULATED 8NM_012247*SEPHS1SELENOPHOSPHATE SYNTHETASE 1NM_012325*MAPRE1MICROTUBULE-ASSOCIATED PROTEIN, RP / EBFAMILY, MEMBER 1NM_015417*SPEF1CHROMOSOME 20 OPEN READING FRAME 28NM_016058*TPRKBTP53RK BINDING PROTEINNM_018014*BCL11AB-CELL CLL / LYMPHOMA 11A (ZINC FINGERPROTEIN)NM_022754*SFXN1LIKELY ORTHOLOG OF MOUSE SIDEROFLEXIN 1NM_030662*MAP2K2MITOGEN-ACTIVATED PROTEIN KINASE KINASE 2NM_032141*CCDC55COILED-COIL DOMAIN CONTAINING 55NM_130439*MXI1MAX INTERACTOR 1NM_138559*BCL11AB-CELL CLL / LYMPHOMA 11A (ZINC FINGERPROTEIN)NM_175907*ZADH2HYPOTHETICAL PROTEIN BC010734NM_212535*PRKCB1PROTEIN KINASE C, BETA 1FAT10NM_005737ARL4CADP-RIBOSYLATION FACTOR-LIKE 4CBC013648EFHD2EF-HAND DOMAIN FAMILY, MEMBER D2BC031247CCDC67COILED-COIL DOMAIN CONTAINING 67NM_015621CCDC69COILED-COIL DOMAIN CONTAINING 69NM_024099C11ORF48CHROMOSOME 11 OPEN READING FRAME 48NM_016951CKLFCHEMOKINE-LIKE FACTORBC008919TBC1D9BKIAA0676 PROTEINNM_032855HSH2DHEMATOPOIETIC SH2 DOMAIN CONTAININGNM_152788ANKS1BANKYRIN REPEAT AND STERILE ALPHA MOTIFDOMAIN CONTAINING 1BNM_001277CHKACHOLINE KINASE ALPHANM_152434CWF19L2CWF19-LIKE 2, CELL CYCLE CONTROL (S. POMBE)NM_004811LPXNLEUPAXINNM_182739NDUFB6NADH DEHYDROGENASE (UBIQUINONE) 1 BETASUBCOMPLEX, 6, 17 KDABC053602C15ORF38HYPOTHETICAL PROTEIN FLJ35955NM_018976SLC38A2SOLUTE CARRIER FAMILY 38, MEMBER 2BC004967UBAC1UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1BC010360LMBRD1LMBR1 DOMAIN CONTAINING 1BC016381NAHYPOTHETICAL PROTEINBC017101POMZP3POM (POM121 HOMOLOG, RAT) AND ZP3 FUSIONBC026175ATF2ACTIVATING TRANSCRIPTION FACTOR 2BC062359C8ORF47CHROMOSOME 8 OPEN READING FRAME 47NM_000301PLGPLASMINOGENNM_002815PSMD11PROTEASOME (PROSOME, MACROPAIN) 26SSUBUNIT, NON-ATPASE, 11NM_002854PVALBPARVALBUMINNM_012198GCAGRANCALCIN, EF-HAND CALCIUM BINDINGPROTEINNM_017727FLJ20254HYPOTHETICAL PROTEIN FLJ20254NM_021925C2ORF43HYPOTHETICAL PROTEIN FLJ21820NM_138785C6ORF72CHROMOSOME 6 OPEN READING FRAME 72NM_144686TMC4TRANSMEMBRANE CHANNEL-LIKE 4NM_012416RANBP6RAN BINDING PROTEIN 6NM_006899IDH3BISOCITRATE DEHYDROGENASE 3 (NAD+) BETABC001726NOL11NUCLEOLAR PROTEIN 11BC015219RBCK1CHROMOSOME 20 OPEN READING FRAME 18BC034801ZDHHC19ZINC FINGER, DHHC-TYPE CONTAINING 19BC022244PYCR1PYRROLINE-5-CARBOXYLATE REDUCTASE 1NM_006399BATFBASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR,ATF-LIKEBC014949DHX58LIKELY ORTHOLOG OF MOUSE D11LGP2NM_014182ORMDL2ORM1-LIKE 2 (S. CEREVISIAE)NM_024114TRIM48TRIPARTITE MOTIF-CONTAINING 48NM_006607PTTG2PITUITARY TUMOR-TRANSFORMING 2NM_004357CD151CD151 ANTIGEN (RAPH BLOOD GROUP)NM_005513GTF2E1GENERAL TRANSCRIPTION FACTOR IIE,POLYPEPTIDE 1, ALPHA 56 KDANM_016231NLKNEMO-LIKE KINASENM_054033FKBP1BFK506 BINDING PROTEIN 1B, 12.6 KDANM_152646hypothetical protein MGC23270NM_173518C8ORF45CHROMOSOME 8 OPEN READING FRAME 45NM_177951PPM1APROTEIN PHOSPHATASE 1A (FORMERLY 2C),MAGNESIUM-DEPENDENT, ALPHA ISOFORMNM_020990CKMT1BCREATINE KINASE, MITOCHONDRIAL 1BNM_001258CDK3CYCLIN-DEPENDENT KINASE 3NM_138565CTTNCORTACTINNM_018189DPPA4DEVELOPMENTAL PLURIPOTENCY ASSOCIATED 4NM_001330CTF1CARDIOTROPHIN 1BC029541LETM2LEUCINE ZIPPER-EF-HAND CONTAININGTRANSMEMBRANE PROTEIN 2NM_144594GTSF1FAMILY WITH SEQUENCE SIMILARITY 112,MEMBER BNM_173192KCNIP2KV CHANNEL INTERACTING PROTEIN 2BC034468FLJ11171HYPOTHETICAL PROTEIN FLJ11171NM_033306CASP4CASPASE 4, APOPTOSIS-RELATED CYSTEINEPEPTIDASEBC041132KIFC3KINESIN FAMILY MEMBER C3BC011461MITFMICROPHTHALMIA-ASSOCIATED TRANSCRIPTIONFACTORBC046214MPHOSPH8M-PHASE PHOSPHOPROTEIN, MPP8BC057774RG9MTD3RNA (GUANINE-9-) METHYLTRANSFERASE DOMAINCONTAINING 3NM_016606REEP2RECEPTOR ACCESSORY PROTEIN 2NM_145265CCDC127SIMILAR TO RIKEN CDNA 0610011N22BC015056ACAD10ACYL-COENZYME A DEHYDROGENASE FAMILY,MEMBER 10BC007224GALNT10UDP-N-ACETYL-ALPHA-D-GALACTOSAMINE: POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE 10(GALNAC-T10)BC009289ACSBG1ACYL-COA SYNTHETASE BUBBLEGUM FAMILYMEMBER 1BC011786NACHROMOSOME 11 OPEN READING FRAME 43NM_000559HBG2HEMOGLOBIN, GAMMA ANM_024680E2F8E2F TRANSCRIPTION FACTOR 8BC000557PEMTPHOSPHATIDYLETHANOLAMINE N-METHYLTRANSFERASEBC005974VAMP4VESICLE-ASSOCIATED MEMBRANE PROTEIN 4BC009771BCCIPCDK INHIBITOR P21 BINDING PROTEINBC053508ARL6IP2ADP-RIBOSYLATION FACTOR-LIKE 6 INTERACTINGPROTEIN 2NM_001307CLDN7CLAUDIN 7NM_00268812:00 AMSEPTIN 5NM_004123GIPGASTRIC INHIBITORY POLYPEPTIDENM_004545NDUFB1NADH DEHYDROGENASE (UBIQUINONE) 1 BETASUBCOMPLEX, 1, 7 KDANM_004712HGSHEPATOCYTE GROWTH FACTOR-REGULATEDTYROSINE KINASE SUBSTRATENM_005621S100A12S100 CALCIUM BINDING PROTEIN A12(CALGRANULIN C)NM_016388TRAT1T CELL RECEPTOR ASSOCIATED TRANSMEMBRANEADAPTOR 1NM_138998DDX39DEAD (ASP-GLU-ALA-ASP) (SEQ ID NO: 2) BOXPOLYPEPTIDE 39NM_144673CMTM2CKLF-LIKE MARVEL TRANSMEMBRANE DOMAINCONTAINING 2NM_182597C7ORF53HYPOTHETICAL PROTEIN FLJ39575BC035601WWC3KIAA1280 PROTEINBC036365C10ORF81HYPOTHETICAL PROTEIN LOC338564NM_002103GYS1GLYCOGEN SYNTHASE 1 (MUSCLE)NM_145252LOC124220SIMILAR TO COMMON SALIVARY PROTEIN 1NM_139280ORMDL3HYPOTHETICAL PROTEIN LOC51242NM_022372GBLG PROTEIN BETA SUBUNIT-LIKEBC052805EPB49ERYTHROCYTE MEMBRANE PROTEIN BAND 4.9(DEMATIN)NM_014551NCAPH2KLEISIN BETANM_017848FAM120CCHROMOSOME X OPEN READING FRAME 17BC008141UCHL5IPTHREE PRIME REPAIR EXONUCLEASE 2NM_005832KCNMB2POTASSIUM LARGE CONDUCTANCE CALCIUM-ACTIVATED CHANNEL, SUBFAMILY M, BETAMEMBER 2NM_173517VKORC1L1VITAMIN K EPOXIDE REDUCTASE COMPLEX,SUBUNIT 1-LIKE 1NM_173473C10ORF104CHROMOSOME 10 OPEN READING FRAME 104NM_030650KIAA1715KIAA1715NM_014570ARFGAP3ADP-RIBOSYLATION FACTOR GTPASE ACTIVATINGPROTEIN 3NM_021159RAP1GDS1RAP1, GTP-GDP DISSOCIATION STIMULATOR 1BC017066PRRC1HYPOTHETICAL PROTEIN MGC12103NM_014805EPM2AIP1EPM2A (LAFORIN) INTERACTING PROTEIN 1BC033734C17ORF66CHROMOSOME 17 OPEN READING FRAME 66NM_021644HNRPH3HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEINH3 (2H9)BC021987NMIN-MYC (AND STAT) INTERACTORNM_002489NDUFA4NADH DEHYDROGENASE (UBIQUINONE) 1 ALPHASUBCOMPLEX, 4, 9 KDANM_033542DBNDD2CHROMOSOME 20 OPEN READING FRAME 35BC015754CADPSCA2+-DEPENDENT SECRETION ACTIVATORNM_032357CCDC115HYPOTHETICAL PROTEIN MGC12981XM_291436BC012266ATG12ATG12 AUTOPHAGY RELATED 12 HOMOLOG (S. CEREVISIAE)BC012377EGFL7EGF-LIKE-DOMAIN, MULTIPLE 7BC017943PPP1R1CPROTEIN PHOSPHATASE 1, REGULATORY(INHIBITOR) SUBUNIT 1CBC058031HPHAPTOGLOBINBC060828ARID3AAT RICH INTERACTIVE DOMAIN 3A (BRIGHT-LIKE)NM_144586LYPD1LY6 / PLAUR DOMAIN CONTAINING 1BC009106SEC16BLEUCINE ZIPPER TRANSCRIPTION REGULATOR 2NM_018990CXORF9CHROMOSOME X OPEN READING FRAME 9NM_004935CDK5CYCLIN-DEPENDENT KINASE 5BC014484TOR1ATORSIN FAMILY 1, MEMBER A (TORSIN A)BC063111GGT6GAMMA-GLUTAMYLTRANSFERASE 6 HOMOLOG(RAT)NM_023937MRPL34MITOCHONDRIAL RIBOSOMAL PROTEIN L34NM_030810TXNDC5THIOREDOXIN DOMAIN CONTAINING 5NM_138463TLCD1TLC DOMAIN CONTAINING 1BC007919STARD10START DOMAIN CONTAINING 10BC016703ACSM5HYPOTHETICAL PROTEIN FLJ20581NM_001004354NRARPSIMILAR TO ANKYRIN-REPEAT PROTEIN NRARPNM_002436MPP1MEMBRANE PROTEIN, PALMITOYLATED 1, 55 KDANM_004013DMDDYSTROPHIN (MUSCULAR DYSTROPHY, DUCHENNEAND BECKER TYPES)NM_018335C14ORF131CHROMOSOME 14 OPEN READING FRAME 131NM_138385TMEM129TRANSMEMBRANE PROTEIN 129NM_001823CKBCREATINE KINASE, BRAINNM_004440EPHA7EPH RECEPTOR A7NM_006779CDC42EP2CDC42 EFFECTOR PROTEIN (RHO GTPASE BINDING) 2NM_007162TFEBTRANSCRIPTION FACTOR EBNM_014248RBX1RING-BOX 1NM_016267VGLL1VESTIGIAL LIKE 1 (DROSOPHILA)NM_181656C17ORF58CHROMOSOME 17 OPEN READING FRAME 58NM_138482hypothetical protein BC009264BC026345KIAA1189KIAA1189NM_032315SLC25A33PNC1 PROTEINNM_002944ROS1V-ROS UR2 SARCOMA VIRUS ONCOGENE HOMOLOG1 (AVIAN)BC017048GJB2GAP JUNCTION PROTEIN, BETA 2, 26 KDA(CONNEXIN 26)BC039814ZRANB2ZINC FINGER PROTEIN 265NM_001044SLC6A3SOLUTE CARRIER FAMILY 6 (NEUROTRANSMITTERTRANSPORTER, DOPAMINE), MEMBER 3NM_138470hypothetical protein BC008131NM_005084PLA2G7PHOSPHOLIPASE A2, GROUP VII (PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE,PLASMA)BC012499SIRT1SIRTUIN (SILENT MATING TYPE INFORMATIONREGULATION 2 HOMOLOG) 1 (S. CEREVISIAE)BC045532LSM8LSM8 HOMOLOG, U6 SMALL NUCLEAR RNAASSOCIATED (S. CEREVISIAE)NM_003295TPT1TUMOR PROTEIN, TRANSLATIONALLY-CONTROLLED 1NM_006912RIT1RAS-LIKE WITHOUT CAAX 1NM_014184CNIH4CORNICHON HOMOLOG 4 (DROSOPHILA)BC003065CDK2CYCLIN-DEPENDENT KINASE 2BC009793ERCC8EXCISION REPAIR CROSS-COMPLEMENTINGRODENT REPAIR DEFICIENCY, COMPLEMENTATIONGROUP 8NM_005114HS3ST1HEPARAN SULFATE (GLUCOSAMINE) 3-O-SULFOTRANSFERASE 1NM_018129PNPOPYRIDOXINE 5′-PHOSPHATE OXIDASENM_152285ARRDC1ARRESTIN DOMAIN CONTAINING 1BC009710GOSR2GOLGI SNAP RECEPTOR COMPLEX MEMBER 2NM_015966ERGIC3ERGIC AND GOLGI 3NM_020370GPR84G PROTEIN-COUPLED RECEPTOR 84NM_130398EXO1EXONUCLEASE 1NM_145865ANKS4BANKYRIN REPEAT AND STERILE ALPHA MOTIFDOMAIN CONTAINING 4BBC001234LOH11CR2ALOSS OF HETEROZYGOSITY, 11, CHROMOSOMALREGION 2, GENE ABC062625SLC39A4SOLUTE CARRIER FAMILY 39 (ZINC TRANSPORTER),MEMBER 4BC001889NAPGN-ETHYLMALEIMIDE-SENSITIVE FACTORATTACHMENT PROTEIN, GAMMABC013768PCCBPROPIONYL COENZYME A CARBOXYLASE, BETAPOLYPEPTIDEBC020651MRPL35MITOCHONDRIAL RIBOSOMAL PROTEIN L35BC051291RDH11RETINOL DEHYDROGENASE 11 (ALL-TRANS AND 9-CIS)BC069328BMFBCL2 MODIFYING FACTORNM_006426DPYSL4DIHYDROPYRIMIDINASE-LIKE 4NM_178863KCTD13POTASSIUM CHANNEL TETRAMERISATION DOMAINCONTAINING 13BC004176SSH3SLINGSHOT HOMOLOG 3 (DROSOPHILA)BC008790GSTM3GLUTATHIONE S-TRANSFERASE M3 (BRAIN)BC010176NY-SAR-48SARCOMA ANTIGEN NY-SAR-48BC020885C12ORF65HYPOTHETICAL PROTEIN FLJ38663BC034554SERPINA3SERPIN PEPTIDASE INHIBITOR, CLADE A (ALPHA-1ANTIPROTEINASE, ANTITRYPSIN), MEMBER 3NM_000394CRYAACRYSTALLIN, ALPHA ANM_078476BTN2A1BUTYROPHILIN, SUBFAMILY 2, MEMBER A1BC015904MRPL10MITOCHONDRIAL RIBOSOMAL PROTEIN L10BC019039RGS3REGULATOR OF G-PROTEIN SIGNALLING 3BC067445DAB1DISABLED HOMOLOG 1 (DROSOPHILA)NM_003221TFAP2BTRANSCRIPTION FACTOR AP-2 BETA (ACTIVATINGENHANCER BINDING PROTEIN 2 BETA)NM_015959TXNDC14THIOREDOXIN DOMAIN CONTAINING 14BC010033QPRTQUINOLINATE PHOSPHORIBOSYLTRANSFERASE(NICOTINATE-NUCLEOTIDE PYROPHOSPHORYLASE(CARBOXYLATING))NM_152522ARL6IP6ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTINGPROTEIN 6BC019254ENOX2CYTOSOLIC OVARIAN CARCINOMA ANTIGEN 1NM_012148DUX3DOUBLE HOMEOBOX, 3NM_025004CCDC15COILED-COIL DOMAIN CONTAINING 15BC017475TTC15TETRATRICOPEPTIDE REPEAT DOMAIN 15NM_172211CSF1COLONY STIMULATING FACTOR 1 (MACROPHAGE)BC007862GPR108G PROTEIN-COUPLED RECEPTOR 108BC010850HEATR2HYPOTHETICAL PROTEIN FLJ20397NM_016009SH3GLB1SH3-DOMAIN GRB2-LIKE ENDOPHILIN B1NM_152328ADSSL1ADENYLOSUCCINATE SYNTHASE LIKE 1BC020867SLC6A13SOLUTE CARRIER FAMILY 6 (NEUROTRANSMITTERTRANSPORTER, GABA), MEMBER 13NM_178126FAM134CHYPOTHETICAL PROTEIN LOC162427NM_007241SNF8SNF8, ESCRT-II COMPLEX SUBUNIT, HOMOLOG (S. CEREVISIAE)NM_016440VRK3VACCINIA RELATED KINASE 3BC035314BXDC1BRIX DOMAIN CONTAINING 1NM_030881DDX17DEAD (ASP-GLU-ALA-ASP) BOX POLYPEPTIDE 17NM_001033578SGK3SERUM / GLUCOCORTICOID REGULATED KINASEFAMILY, MEMBER 3BC010155FDX1LSIMILAR TO RIKEN CDNA B230118G17 GENENM_018667SMPD3SPHINGOMYELIN PHOSPHODIESTERASE 3,NEUTRAL MEMBRANE (NEUTRALSPHINGOMYELINASE II)NM_017812CHCHD3COILED-COIL-HELIX-COILED-COIL-HELIX DOMAINCONTAINING 3NM_001613ACTG2ACTIN, ALPHA 2, SMOOTH MUSCLE, AORTABC031329TMEM149U2(RNU2) SMALL NUCLEAR RNA AUXILIARYFACTOR 1-LIKE 4BC039256PDS5BANDROGEN-INDUCED PROLIFERATION INHIBITORNM_017634KCTD9POTASSIUM CHANNEL TETRAMERISATION DOMAINCONTAINING 9NM_001017980LOC203547HYPOTHETICAL PROTEIN LOC203547BC053320CTBP1C-TERMINAL BINDING PROTEIN 1NM_152619DCLK2DOUBLECORTIN AND CAM KINASE-LIKE 2BC033668ARHGAP28KIAA1314 PROTEINBC059396FAM92A3FAMILY WITH SEQUENCE SIMILARITY 92, MEMBERA3NM_080660ZC3HAV1LSIMILAR TO RIKEN CDNA 1200014N16 GENEBC003551TGM2TRANSGLUTAMINASE 2 (C POLYPEPTIDE, PROTEIN-GLUTAMINE-GAMMA-GLUTAMYLTRANSFERASE)NM_172341LIN37PRESENILIN ENHANCER 2 HOMOLOG (C. ELEGANS)NM_005158ABL2V-ABL ABELSON MURINE LEUKEMIA VIRALONCOGENE HOMOLOG 2 (ARG, ABELSON-RELATEDGENE)NM_005558LAD1LADININ 1NM_000624SERPINA5SERPIN PEPTIDASE INHIBITOR, CLADE A (ALPHA-1ANTIPROTEINASE, ANTITRYPSIN), MEMBER 5NM_173799VSTM3V-SET AND IMMUNOGLOBULIN DOMAINCONTAINING 9NM_003592CUL1CULLIN 1BC017594APIPAPAF1 INTERACTING PROTEINNM_032498RHOXF2PEPP SUBFAMILY GENE 2BC008730HK1HEXOKINASE 1BC016276DLG7DISCS, LARGE HOMOLOG 7 (DROSOPHILA)BC033708RALGPS1RAL GEF WITH PH DOMAIN AND SH3 BINDINGMOTIF 1BC051000TCL1BT-CELL LEUKEMIA / LYMPHOMA 1BBC066974NAHYPOTHETICAL PROTEINBC022983LNX1LIGAND OF NUMB-PROTEIN X 1NM_003256TIMP4TIMP METALLOPEPTIDASE INHIBITOR 4NM_003674CDK10CYCLIN-DEPENDENT KINASE (CDC2-LIKE) 10BC004549DUS3LDIHYDROURIDINE SYNTHASE 3-LIKE (S. CEREVISIAE)BC015596*C21ORF51CHROMOSOME 21 OPEN READING FRAME 51BC018206*FAM128BHYPOTHETICAL PROTEIN FLJ14346BC018722*ASPSCR1ALVEOLAR SOFT PART SARCOMA CHROMOSOMEREGION, CANDIDATE 1BC022357*RPL17RIBOSOMAL PROTEIN L17BC023152*GYG2GLYCOGENIN 2BC025700*AFF4AF4 / FMR2 FAMILY, MEMBER 4BC032825*SH3GL2SH3-DOMAIN GRB2-LIKE 2BC038838*PRR16MESENCHYMAL STEM CELL PROTEIN DSC54BC052805*EPB49ERYTHROCYTE MEMBRANE PROTEIN BAND 4.9(DEMATIN)BC056415*RPAP3HYPOTHETICAL PROTEIN FLJ21908BC065370*C20ORF112CHROMOSOME 20 OPEN READING FRAME 112NM_001032296*STK24SERINE / THREONINE KINASE 24 (STE20 HOMOLOG,YEAST)NM_002498*NEK3NIMA (NEVER IN MITOSIS GENE A)-RELATEDKINASE 3NM_002624*PFDN5PREFOLDIN SUBUNIT 5NM_004329*BMPR1ABONE MORPHOGENETIC PROTEIN RECEPTOR, TYPEIANM_014245*RNF7RING FINGER PROTEIN 7NM_014548*TMOD2TROPOMODULIN 2 (NEURONAL)NM_015646*RAP1BRAP1B, MEMBER OF RAS ONCOGENE FAMILYNM_017949*CUEDC1CUE DOMAIN CONTAINING 1NM_018393*TCP11L1T-COMPLEX 11 (MOUSE) LIKE 1NM_018679*TCP11T-COMPLEX 11 (MOUSE)NM_024591*CHMP6CHROMATIN MODIFYING PROTEIN 6NM_032368*LZICLEUCINE ZIPPER AND CTNNBIP1 DOMAINCONTAININGNM_033118*MYLK2MYOSIN LIGHT CHAIN KINASE 2, SKELETALMUSCLENM_130807*MOBKL2AMOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE2A (YEAST)NM_145173*DIRAS1DIRAS FAMILY, GTP-BINDING RAS-LIKE 1NM_152376*UBXD3UBX DOMAIN CONTAINING 3NM_182493*MLCKMLCK PROTEINBC056907*NANASUMO1BC033766NDUFV3NADH DEHYDROGENASE (UBIQUINONE)FLAVOPROTEIN 3, 10 KDANM_001312CRIP2CYSTEINE-RICH PROTEIN 2NM_004111FEN1FLAP STRUCTURE-SPECIFIC ENDONUCLEASE 1NM_000805GASTGASTRINNM_030645SH3BP5LSH3-BINDING DOMAIN PROTEIN 5-LIKEBC019337IGHG1IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 1(G1M MARKER)BC056673PPP1R2P9PROTEIN PHOSPHATASE 1, REGULATORY(INHIBITOR) SUBUNIT 2 PSEUDOGENE 9BC054520MEF2DMADS BOX TRANSCRIPTION ENHANCER FACTOR 2,POLYPEPTIDE D (MYOCYTE ENHANCER FACTOR2D)NM_006902PRRX1PAIRED RELATED HOMEOBOX 1NM_004436ENSAENDOSULFINE ALPHANM_006255PRKCHPROTEIN KINASE C, ETANM_007080LSM6LSM6 HOMOLOG, U6 SMALL NUCLEAR RNAASSOCIATED (S. CEREVISIAE)NM_000860HPGDHYDROXYPROSTAGLANDIN DEHYDROGENASE 15-(NAD)NM_144679C17ORF56CHROMOSOME 17 OPEN READING FRAME 56NM_017431PRKAG3PROTEIN KINASE, AMP-ACTIVATED, GAMMA 3NON-CATALYTIC SUBUNITNM_031473IFT81INTRAFLAGELLAR TRANSPORT 81 HOMOLOG(CHLAMYDOMONAS)BC064593DCP2DCP2 DECAPPING ENZYME HOMOLOG (S. CEREVISIAE)BC007347CHD2CHROMODOMAIN HELICASE DNA BINDINGPROTEIN 2BC003690IPO4IMPORTIN 4BC016327NUP62CLHYPOTHETICAL PROTEIN FLJ20130NM_080600MAGMYELIN ASSOCIATED GLYCOPROTEINBC017258MCM2MCM2 MINICHROMOSOME MAINTENANCEDEFICIENT 2, MITOTIN (S. CEREVISIAE)NM_017785CCDC99HYPOTHETICAL PROTEIN FLJ20364BC000809TCEAL1TRANSCRIPTION ELONGATION FACTOR A (SII)-LIKE 1NM_000485APRTADENINE PHOSPHORIBOSYLTRANSFERASENM_138820HIGD2AHIG1 DOMAIN FAMILY, MEMBER 2ABC009415KIF26AKINESIN FAMILY MEMBER 26ABC017440TRAPPC2LHEMATOPOIETIC STEM / PROGENITOR CELLS 176NM_001092ABRACTIVE BCR-RELATED GENEBC013352HTF9CHPAII TINY FRAGMENTS LOCUS 9CNM_021947SRRSERINE RACEMASEBC011585PRKCDBPPROTEIN KINASE C, DELTA BINDING PROTEINBC052600ZNF718ZINC FINGER PROTEIN 718BC004518SYT17SYNAPTOTAGMIN XVIINM_178509STXBP4SYNTAXIN BINDING PROTEIN 4BC017770NANABC066938DDX43DEAD (ASP-GLU-ALA-ASP) (SEQ ID NO: 2) BOXPOLYPEPTIDE 43BC000393FAM127BDKFZP564B147 PROTEINBC025787ALKBH1ALKB, ALKYLATION REPAIR HOMOLOG 1 (E. COLI)BC015944TIA1TIA1 CYTOTOXIC GRANULE-ASSOCIATED RNABINDING PROTEINNM_017988SCYL2SCY1-LIKE 2 (S. CEREVISIAE)NM_002020FLT4FMS-RELATED TYROSINE KINASE 4NM_031472TRPT1TRNA PHOSPHOTRANSFERASE 1BC001728*TFPTTCF3 (E2A) FUSION PARTNER (IN CHILDHOODLEUKEMIA)BC003566*ZNF24ZINC FINGER PROTEIN 24 (KOX 17)BC005383*CETN3CENTRIN, EF-HAND PROTEIN, 3 (CDC31 HOMOLOG,YEAST)BC007048*ZMYM5ZINC FINGER, MYM-TYPE 5BC010125*C3ORF37CHROMOSOME 3 OPEN READING FRAME 37BC011804*C1ORF165CHROMOSOME 1 OPEN READING FRAME 165BC015803*IRF2INTERFERON REGULATORY FACTOR 2BC017314*ETS1V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENEHOMOLOG 1 (AVIAN)BC036335*BTBD12BTB (POZ) DOMAIN CONTAINING 12BC036572*ZCCHC12ZINC FINGER, CCHC DOMAIN CONTAINING 12BC051688*FLJ10781HYPOTHETICAL PROTEIN FLJ10781BC056402*LOC144097HYPOTHETICAL PROTEIN BC007540BC067299*MDM4MDM4, TRANSFORMED 3T3 CELL DOUBLE MINUTE4, P53 BINDING PROTEIN (MOUSE)NM_000176*NR3C1NUCLEAR RECEPTOR SUBFAMILY 3, GROUP C,MEMBER 1 (GLUCOCORTICOID RECEPTOR)NM_001008239*C18ORF25CHROMOSOME 18 OPEN READING FRAME 25NM_001722*POLR3DPOLYMERASE (RNA) III (DNA DIRECTED)POLYPEPTIDE D, 44 KDANM_001895*CSNK2A1CASEIN KINASE 2, ALPHA 1 POLYPEPTIDENM_002739*PRKCGPROTEIN KINASE C, GAMMANM_002938*RNF4RING FINGER PROTEIN 4NM_003141*TRIM21TRIPARTITE MOTIF-CONTAINING 21NM_003345*UBE2IUBIQUITIN-CONJUGATING ENZYME E2I (UBC9HOMOLOG, YEAST)NM_003352*SUMO1SMT3 SUPPRESSOR OF MIF TWO 3 HOMOLOG 1(YEAST)NM_004454*ETV5ETS VARIANT GENE 5 (ETS-RELATED MOLECULE)NM_006977*ZBTB25ZINC FINGER AND BTB DOMAIN CONTAINING 25NM_014720*SLKSTE20-LIKE KINASE (YEAST)NM_032141*CCDC55COILED-COIL DOMAIN CONTAINING 55NM_145796*POGZPOGO TRANSPOSABLE ELEMENT WITH ZNFDOMAINNM_175907*ZADH2HYPOTHETICAL PROTEIN BC010734NM_212540*E2F6E2F TRANSCRIPTION FACTOR 6UFM1NM_005879TRAIPTRAF INTERACTING PROTEINNM_001018RPS15RIBOSOMAL PROTEIN S15NM_013974DDAH2DIMETHYLARGININEDIMETHYLAMINOHYDROLASE 2NM_001278CHUKCONSERVED HELIX-LOOP-HELIX UBIQUITOUSKINASEBC012611EIF4EEUKARYOTIC TRANSLATION INITIATION FACTOR4ENM_006819STIP1STRESS-INDUCED-PHOSPHOPROTEIN 1(HSP70 / HSP90-ORGANIZING PROTEIN)NM_024647NUP43NUCLEOPORIN 43 KDANM_007045FGFR1OPFGFR1 ONCOGENE PARTNERNM_014460CSDC2COLD SHOCK DOMAIN CONTAINING C2, RNABINDINGNM_021260ZFYVE1ZINC FINGER, FYVE DOMAIN CONTAINING 1NM_017437CPSF2CLEAVAGE AND POLYADENYLATION SPECIFICFACTOR 2, 100 KDANM_138722BCL2L14BCL2-LIKE 14 (APOPTOSIS FACILITATOR)NM_016059PPIL1PEPTIDYLPROLYL ISOMERASE (CYCLOPHILIN)-LIKE 1NM_020139BDH23-HYDROXYBUTYRATE DEHYDROGENASE, TYPE 2NM_182493MLCKMLCK PROTEINBC000578HPRT1HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE 1(LESCH-NYHAN SYNDROME)BC060785TRIM40TRIPARTITE MOTIF-CONTAINING 40BC003132NUDCNUCLEAR DISTRIBUTION GENE C HOMOLOG (A. NIDULANS)NM_031219HDHD3HALOACID DEHALOGENASE-LIKE HYDROLASEDOMAIN CONTAINING 3NM_002358MAD2L1MAD2 MITOTIC ARREST DEFICIENT-LIKE 1 (YEAST)NM_006578GNB5GUANINE NUCLEOTIDE BINDING PROTEIN (GPROTEIN), BETA 5NM_004064CDKN1BCYCLIN-DEPENDENT KINASE INHIBITOR 1B (P27,KIP1)BC030280KIAA0513KIAA0513NM_005338HIP1HUNTINGTIN INTERACTING PROTEIN 1NM_004881TP53I3TUMOR PROTEIN P53 INDUCIBLE PROTEIN 3BC015395CCDC148HYPOTHETICAL PROTEIN BC015395NM_000394CRYAACRYSTALLIN, ALPHA ABC005955C8ORF53CHROMOSOME 8 OPEN READING FRAME 53BC001327IFRD2INTERFERON-RELATED DEVELOPMENTALREGULATOR 2BC021551NFATC2IPNUCLEAR FACTOR OF ACTIVATED T-CELLS,CYTOPLASMIC, CALCINEURIN-DEPENDENT 2INTERACTING PROTEINBC050537FLJ20160FLJ20160 PROTEINBC058862TSKSTESTIS-SPECIFIC KINASE SUBSTRATENM_005235ERBB4V-ERB-A ERYTHROBLASTIC LEUKEMIA VIRALONCOGENE HOMOLOG 4 (AVIAN)NM_014012REM1RAS (RAD AND GEM)-LIKE GTP-BINDING 1NM_022110FKBPLFK506 BINDING PROTEIN LIKENM_006147IRF6INTERFERON REGULATORY FACTOR 6NM_001349DARSASPARTYL-TRNA SYNTHETASEBC064945SCYL1BP1SCY1-LIKE 1 BINDING PROTEIN 1NM_032385C5ORF4CHROMOSOME 5 OPEN READING FRAME 4NM_172037RDH10RETINOL DEHYDROGENASE 10 (ALL-TRANS)NM_173621C17ORF44CHROMOSOME 17 OPEN READING FRAME 44NM_004074COX8ACYTOCHROME C OXIDASE SUBUNIT 8A(UBIQUITOUS)NM_022156DUS1LDIHYDROURIDINE SYNTHASE 1-LIKE (S. CEREVISIAE)NM_016401C11ORF73HYPOTHETICAL PROTEIN HSPC138NM_019617GKN1GASTROKINE 1BC054501DNM2DYNAMIN 2NM_058173MUCL1SMALL BREAST EPITHELIAL MUCINBC032307CCDC123HYPOTHETICAL PROTEIN FLJ14640BC034028SHARPINSHANK-ASSOCIATED RH DOMAIN INTERACTORBC015202CENPTCHROMOSOME 16 OPEN READING FRAME 56BC013957FAM62BFAMILY WITH SEQUENCE SIMILARITY 62 (C2DOMAIN CONTAINING) MEMBER BBC015569ARL6IP4ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTINGPROTEIN 4BC020221STACSH3 AND CYSTEINE RICH DOMAINBC053895IRS1INSULIN RECEPTOR SUBSTRATE 1NM_002748MAPK6MITOGEN-ACTIVATED PROTEIN KINASE 6NM_198086JUBJUB, AJUBA HOMOLOG (XENOPUS LAEVIS)NM_006621AHCYL1S-ADENOSYLHOMOCYSTEINE HYDROLASE-LIKE 1NM_018698NXT2NUCLEAR TRANSPORT FACTOR 2-LIKE EXPORTFACTOR 2NM_005034POLR2KPOLYMERASE (RNA) II (DNA DIRECTED)POLYPEPTIDE K, 7.0 KDANM_018438FBXO6F-BOX PROTEIN 6NM_033547INTS4INTEGRATOR COMPLEX SUBUNIT 4NM_153212GJB4GAP JUNCTION PROTEIN, BETA 4 (CONNEXIN 30.3)NM_175738RAB37RAB37, MEMBER RAS ONCOGENE FAMILYBC013031PHLDB1PLECKSTRIN HOMOLOGY-LIKE DOMAIN, FAMILY B,MEMBER 1NM_001005465OR10G3OLFACTORY RECEPTOR, FAMILY 10, SUBFAMILY G,MEMBER 3NM_001899CST4CYSTATIN SNM_004753DHRS3DEHYDROGENASE / REDUCTASE (SDR FAMILY)MEMBER 3NM_021992TMSL8THYMOSIN-LIKE 8NM_197970BOLLBOL, BOULE-LIKE (DROSOPHILA)NM_139246C9ORF97CHROMOSOME 9 OPEN READING FRAME 97NM_005586MDFIMYOD FAMILY INHIBITORBC041831TLE3TRANSDUCIN-LIKE ENHANCER OF SPLIT 3 (E(SP1)HOMOLOG, DROSOPHILA)NM_003130SRISORCINBC030237SLC22A18ASSOLUTE CARRIER FAMILY 22 (ORGANIC CATIONTRANSPORTER), MEMBER 18 ANTISENSEBC053351DLX1DISTAL-LESS HOMEOBOX 1BC022034LDHAL6BLACTATE DEHYDROGENASE A-LIKE 6BBC031964GLULGLUTAMATE-AMMONIA LIGASE (GLUTAMINESYNTHETASE)NM_032350C7ORF50HYPOTHETICAL PROTEIN MGC11257NM_152646hypothetical protein MGC23270BC024245SALL2SAL-LIKE 2 (DROSOPHILA)NM_001004300ZNF720ZINC FINGER PROTEIN 720NM_079422MYL1MYOSIN, LIGHT POLYPEPTIDE 1, ALKALI;SKELETAL, FASTNM_024295DERL1DER1-LIKE DOMAIN FAMILY, MEMBER 1BC026241UBE3CUBIQUITIN PROTEIN LIGASE E3CBC064144NANANM_152266C19ORF40HYPOTHETICAL PROTEIN MGC32020NM_017722TRMT1TRM1 TRNA METHYLTRANSFERASE 1 HOMOLOG (S. CEREVISIAE)NM_000905NPYNEUROPEPTIDE YBC001553CHMP2BCHROMATIN MODIFYING PROTEIN 2BNM_006438COLEC10COLLECTIN SUB-FAMILY MEMBER 10 (C-TYPELECTIN)NM_014424HSPB7HEAT SHOCK 27 KDA PROTEIN FAMILY, MEMBER 7(CARDIOVASCULAR)NM_001179ART3ADP-RIBOSYLTRANSFERASE 3NM_020348CNNM1CYCLIN M1NM_006928SILVSILVER HOMOLOG (MOUSE)NM_022568ALDH8A1ALDEHYDE DEHYDROGENASE 8 FAMILY, MEMBERA1NM_178152DCXDOUBLECORTEX; LISSENCEPHALY, X-LINKED(DOUBLECORTIN)NM_153822PSMD4PROTEASOME (PROSOME, MACROPAIN) 26SSUBUNIT, NON-ATPASE, 4NM_001699AXLAXL RECEPTOR TYROSINE KINASEBC006195ACLYATP CITRATE LYASENM_020397CAMK1DCALCIUM / CALMODULIN-DEPENDENT PROTEINKINASE IDBC017249ENO3ENOLASE 1, (ALPHA)BC001600CDC123CHROMOSOME 10 OPEN READING FRAME 7NM_024770METTL8HYPOTHETICAL PROTEIN FLJ13984NM_194270MORN2MORN REPEAT CONTAINING 2NM_022650RASA1RAS P21 PROTEIN ACTIVATOR (GTPASEACTIVATING PROTEIN) 1BC005830ANXA9ANNEXIN A9NM_014065ASTE1ASTEROID HOMOLOG 1 (DROSOPHILA)BC014244RTN2RETICULON 2BC024002FNDC8FIBRONECTIN TYPE III DOMAIN CONTAINING 8NM_178034PLA2G4DPHOSPHOLIPASE A2, GROUP IVD (CYTOSOLIC)BC025266TASP1TASPASE, THREONINE ASPARTASE, 1NM_003928FAM127ACAAX BOX 1NM_017819LOC131909RNA (GUANINE-9-) METHYLTRANSFERASE DOMAINCONTAINING 1NM_018158SLC4A1APSOLUTE CARRIER FAMILY 4 (ANION EXCHANGER),MEMBER 1, ADAPTOR PROTEINNM_175571GIMAP8GTPASE, IMAP FAMILY MEMBER 8BC000453PCM1PERICENTRIOLAR MATERIAL 1NM_000910NPY2RNEUROPEPTIDE Y RECEPTOR Y2NM_018679TCP11T-COMPLEX 11 (MOUSE)NM_022559GH1CHORIONIC SOMATOMAMMOTROPIN HORMONE 1(PLACENTAL LACTOGEN)BC030957ANK1ANKYRIN 1, ERYTHROCYTICNM_003168SUPT4H1SUPPRESSOR OF TY 4 HOMOLOG 1 (S. CEREVISIAE)BC012095BST1BONE MARROW STROMAL CELL ANTIGEN 1BC013740SLC2A6SOLUTE CARRIER FAMILY 2 (FACILITATEDGLUCOSE TRANSPORTER), MEMBER 6NM_016505ZCCHC17ZINC FINGER, CCHC DOMAIN CONTAINING 17NM_018697LANCL2LANC LANTIBIOTIC SYNTHETASE COMPONENT C-LIKE 2 (BACTERIAL)NM_152619DCLK2DOUBLECORTIN AND CAM KINASE-LIKE 2NM_152770C4ORF22HYPOTHETICAL PROTEIN MGC35043NM_004401DFFADNA FRAGMENTATION FACTOR, 45 KDA, ALPHAPOLYPEPTIDENM_030636EEPD1KIAA1706 PROTEINBC014260PARP3POLY (ADP-RIBOSE) POLYMERASE FAMILY,MEMBER 3BC009010C6ORF142CHROMOSOME 6 OPEN READING FRAME 142BC047722C2ORF64HYPOTHETICAL PROTEIN MGC52110NM_080873ASB11ANKYRIN REPEAT AND SOCS BOX-CONTAINING 11NM_173547TRIM65TRIPARTITE MOTIF-CONTAINING 65BC041668RIPK3RECEPTOR-INTERACTING SERINE-THREONINEKINASE 3BC033728NANABC048217SPATA5SPERMATOGENESIS ASSOCIATED 5NM_001001852PIM3PIM-3 ONCOGENENM_002904RDBPRD RNA BINDING PROTEINBC030608PODNPODOCANBC023982C5ORF32PUTATIVE NUCLEAR PROTEIN ORF1-FL49NM_133332WHSC1WOLF-HIRSCHHORN SYNDROME CANDIDATE 1NM_004040RHOBRAS HOMOLOG GENE FAMILY, MEMBER BBC033708RALGPS1RAL GEF WITH PH DOMAIN AND SH3 BINDINGMOTIF 1NM_002491NDUFB3NADH DEHYDROGENASE (UBIQUINONE) 1 BETASUBCOMPLEX, 3, 12 KDABC015944TIA1TIA1 CYTOTOXIC GRANULE-ASSOCIATED RNABINDING PROTEINBC050688RPSARIBOSOMAL PROTEIN SANM_002443MSMBMICROSEMINOPROTEIN, BETA-NM_172314IL25INTERLEUKIN 17ENM_019845RPRMREPRIMO, TP53 DEPENDENT G2 ARREST MEDIATORCANDIDATEBC013163DCUN1D1DCN1, DEFECTIVE IN CULLIN NEDDYLATION 1,DOMAIN CONTAINING 1 (S. CEREVISIAE)BC017741GTDC1PRO0159 PROTEINBC023152GYG2GLYCOGENIN 2NM_005663WHSC2WOLF-HIRSCHHORN SYNDROME CANDIDATE 2NM_000214JAG1JAGGED 1 (ALAGILLE SYNDROME)NM_004403DFNA5DEAFNESS, AUTOSOMAL DOMINANT 5NM_022073EGLN3HYPOTHETICAL PROTEIN FLJ21620NM_030571NDFIP1NEDD4 FAMILY INTERACTING PROTEIN 1NM_145252LOC124220SIMILAR TO COMMON SALIVARY PROTEIN 1BC000772SIPA1L3SIGNAL-INDUCED PROLIFERATION-ASSOCIATED 1LIKE 3NM_006579EBPEMOPAMIL BINDING PROTEIN (STEROLISOMERASE)BC014441NSUN4NOL1 / NOP2 / SUN DOMAIN FAMILY, MEMBER 4BC019902CCDC21COILED-COIL DOMAIN CONTAINING 21BC036827LILRB2LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTOR,SUBFAMILY B (WITH TM AND ITIM DOMAINS),MEMBER 2NM_001680FXYD2FXYD DOMAIN CONTAINING ION TRANSPORTREGULATOR 2NM_006439MAB21L2MAB-21-LIKE 2 (C. ELEGANS)NM_032786ZC3H10ZINC FINGER CCCH-TYPE CONTAINING 10NM_024613PLEKHF2PLECKSTRIN HOMOLOGY DOMAIN CONTAINING,FAMILY F (WITH FYVE DOMAIN) MEMBER 2NM_001752CATCATALASENM_152471hypothetical protein MGC17515NM_152716PATL1FLJ36874 PROTEINBC004243BCAT2BRANCHED CHAIN AMINOTRANSFERASE 2,MITOCHONDRIALBC056246GALNT3UDP-N-ACETYL-ALPHA-D-GALACTOSAMINE:POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE 3(GALNAC-T3)NM_022133SNX16SORTING NEXIN 16NM_025221KCNIP4KV CHANNEL INTERACTING PROTEIN 4NM_025234WDR61WD REPEAT DOMAIN 61BC014649GAL3ST1GALACTOSE-3-O-SULFOTRANSFERASE 1NM_002734PRKAR1APROTEIN KINASE, CAMP-DEPENDENT,REGULATORY, TYPE I, ALPHA (TISSUE SPECIFICEXTINGUISHER 1)NM_023934FUNDC2FUN14 DOMAIN CONTAINING 2NM_145173DIRAS1DIRAS FAMILY, GTP-BINDING RAS-LIKE 1NM_020142NDUFA4L2NADH:UBIQUINONE OXIDOREDUCTASE MLRQSUBUNIT HOMOLOGNM_016485VTA1CHROMOSOME 6 OPEN READING FRAME 55NM_000345SNCASYNUCLEIN, ALPHA (NON A4 COMPONENT OFAMYLOID PRECURSOR)BC067447DAB1DISABLED HOMOLOG 1 (DROSOPHILA)NM_001010971SAMD13STERILE ALPHA MOTIF DOMAIN CONTAINING 13BC022043C7ORF36CHROMOSOME 7 OPEN READING FRAME 36BC004233*TTYH2TWEETY HOMOLOG 2 (DROSOPHILA)BC017504*DEF6DIFFERENTIALLY EXPRESSED IN FDCP 6 HOMOLOG(MOUSE)BC018206*FAM128BHYPOTHETICAL PROTEIN FLJ14346BC018404*FGF21FIBROBLAST GROWTH FACTOR 21BC020985*COASYCOENZYME A SYNTHASEBC031469*LOC554207HYPOTHETICAL LOC554207BC058924*UBE2MUBIQUITIN-CONJUGATING ENZYME E2M (UBC12HOMOLOG, YEAST)NM_000020*ACVRL1ACTIVIN A RECEPTOR TYPE II-LIKE 1NM_000154*GALK1GALACTOKINASE 1NM_001014796*DDR2DISCOIDIN DOMAIN RECEPTOR FAMILY, MEMBER 2NM_001105*ACVR1ACTIVIN A RECEPTOR, TYPE INM_001752*CATCATALASENM_002227*JAK1JANUS KINASE 1 (A PROTEIN TYROSINE KINASE)NM_002498*NEK3NIMA (NEVER IN MITOSIS GENE A)-RELATEDKINASE 3NM_002964*S100A8S100 CALCIUM BINDING PROTEIN A8(CALGRANULIN A)NM_003063*SLNSARCOLIPINNM_004972*JAK2JANUS KINASE 2 (A PROTEIN TYROSINE KINASE)NM_005036*PPARAPEROXISOME PROLIFERATIVE ACTIVATEDRECEPTOR, ALPHANM_005122*NR1I3NUCLEAR RECEPTOR SUBFAMILY 1, GROUP I,MEMBER 3NM_005123*NR1H4NUCLEAR RECEPTOR SUBFAMILY 1, GROUP H,MEMBER 4NM_014583*LMCD1LIM AND CYSTEINE-RICH DOMAINS 1NM_015646*RAP1BRAP1B, MEMBER OF RAS ONCOGENE FAMILYNM_016495*TBC1D7TBC1 DOMAIN FAMILY, MEMBER 7NM_021709*SIVA1CD27-BINDING (SIVA) PROTEINNM_030572*C12ORF39CHROMOSOME 12 OPEN READING FRAME 39NM_033360*KRASV-HA-RAS HARVEY RAT SARCOMA VIRALONCOGENE HOMOLOGNM_130807*MOBKL2AMOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE2A (YEAST)NM_145173*DIRAS1DIRAS FAMILY, GTP-BINDING RAS-LIKE 1NM_173541*C10ORF91CHROMOSOME 10 OPEN READING FRAME 91BC004233*NANABC008624*NANAISG15BC013366*URP2UNC-112 RELATED PROTEIN 2BC017314*ETS1V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENEHOMOLOG 1 (AVIAN)BC018404*FGF21FIBROBLAST GROWTH FACTOR 21BC022363*VPS37AVACUOLAR PROTEIN SORTING 37A (YEAST)BC024725*ANKRD50ANKYRIN REPEAT DOMAIN 50BC025307*PRKD2PROTEIN KINASE D2BC029112*SAMSN1SAM DOMAIN, SH3 DOMAIN AND NUCLEARLOCALISATION SIGNALS, 1BC029480*LOC554203HYPOTHETICAL LOC554203BC035636*APBB1IPAMYLOID BETA (A4) PRECURSOR PROTEIN-BINDING, FAMILY B, MEMBER 1 INTERACTINGPROTEINBC038838*PRR16MESENCHYMAL STEM CELL PROTEIN DSC54BC039244*NFYANUCLEAR TRANSCRIPTION FACTOR Y, ALPHABC042999*ASXL2ADDITIONAL SEX COMBS LIKE 2 (DROSOPHILA)BC062423*C7ORF41HYPOTHETICAL PROTEIN ELLS1NM_001571*IRF3INTERFERON REGULATORY FACTOR 3NM_001926*DEFA6DEFENSIN, ALPHA 6, PANETH CELL-SPECIFICNM_002505*NFYANUCLEAR TRANSCRIPTION FACTOR Y, ALPHANM_003141*TRIM21TRIPARTITE MOTIF-CONTAINING 21NM_004304*ALKANAPLASTIC LYMPHOMA KINASE (KI-1)NM_005214*CTLA4CYTOTOXIC T-LYMPHOCYTE-ASSOCIATEDPROTEIN 4NM_005902*SMAD3SMAD, MOTHERS AGAINST DPP HOMOLOG 3(DROSOPHILA)NM_006324*CFDP1CRANIOFACIAL DEVELOPMENT PROTEIN 1NM_007242*DDX19BDEAD (ASP-GLU-ALA-AS) (SEQ ID NO: 2)BOX POLYPEPTIDE 19BNM_012472*LRRC6LEUCINE RICH REPEAT CONTAINING 6NM_015927*TGFB1I1TRANSFORMING GROWTH FACTOR BETA 1INDUCED TRANSCRIPT 1NM_017724*LRRFIP2LEUCINE RICH REPEAT (IN FLII) INTERACTINGPROTEIN 2NM_017855*ODAMAPIN PROTEINNM_023112*OTUB2OTU DOMAIN, UBIQUITIN ALDEHYDE BINDING 2NM_025241*UBXD1UBX DOMAIN CONTAINING 1NM_053283*DCDDERMCIDINNM_172160*KCNAB1POTASSIUM VOLTAGE-GATED CHANNEL, SHAKER-RELATED SUBFAMILY, BETA MEMBER 1NM_175907*ZADH2HYPOTHETICAL PROTEIN BC010734

[0176] The protein targets showing the highest reactivity in a sumo1 PTM assay were the RANBP2 protein, which was previously identified as a sumo1 E3 ligase, and TGFII. In the sumo2 / 3 PTM profile, one of the top reactivities was UbcH9, the only known E2 characterized to date for sumo conjugation. Additionally, among the highest reactivities (top 7) of neddylated proteins were the E2 and E3 enzymes that are known to be involved in the neddylation pathway. The other reactive proteins did not appear to be relevant to the neddylation pathway. Thus, among the top reacting proteins for each of these modifications were the enzymes that are involved in catalysis of the relevant PTM itself. In the case of FAT10, many of the highly reactive proteins were mitotic regulators or cytoskeleton related. To date only one substrate, Mad2, has been described for modification with FAT10, and indeed Mad2 was highly FATtenylated in this assay. FAT10 is known to be highly expressed in certain kinds of cancers, and its overexpression may lead to chromosomal aberrations as well as mitotic arrest. For UFM1 there are no previously known substrates, and therefore all of the identified UFM1 substrates are newly discovered.

[0177] For each of the modifying moieties, signals from the CP-arrested and the CP-released extracts were compared. Two microarrays from each condition were examined, and a two-tailed t-test was used to identify differentially modified proteins. To determine significance, a permutation-based p-value calculation was used, and corrected for false discovery rate (FDR) either using Storey's method or using the Hochberg-Benjamini correction. For each modifying moiety tested (i.e. ubiquitin, sumo1, sumo2 / 3, nedd8, FA10, UFM1, ISG15) the proteins showing significant change in their modification state upon release from the mitotic CP were identified. For each PTM, two biological replicates and two different mitotic conditions (CP-arrested and CP-released) were examined. A subset of the microarray proteins showed a marked difference under the two different conditions but were similar in the biological replicates. These were identified as differentially modified proteins. The data were then clustered based on the differentially modified proteins (FIG. 15). Each row in FIG. 15 represents a different protein that was found to be differentially modified under the two different mitotic conditions. The list of differentially modified proteins was compared for each of the modifications (see Table 7), and the results showed that the proteins were differentially targeted by each of the modifying moieties, and the sets of proteins modified by the different modifying moieties were not overlapping more than would be expected by chance. This is shown in a Venn diagram in FIG. 16, and suggests specialized roles for each different modification in regulating a unique set of target proteins.REFERENCES

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[0272] While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

Claims

1. An assay for determining the post-translational modification (PTM) state of a biological sample from a subject suspected of having a disease or medical conditionin a subject, the method assay comprising the steps of:(i) contacting a functional biological sample comprising proteins at least one protein having PTM enzyme activity with a solid state array, the array comprising an ordered plurality of proteins under conditions that allow post-translational modification (PTM) to occur or that allow PTM to be modified,wherein the PTM is ubiquitination or deubiquitination,wherein the functional biological sample is generated by homogenization of athe biological sample from athe subject suspected of having a disease or medical condition in a detergent-free environment, andwherein the functional biological sample is supplemented with an ATP generating system, andwherein the functional biological sample is a bodily fluid;(ii) identifying post-translationally modified proteins in the solid state array to obtain a PTM state data set that serves as a signature or profile of protein PTMs in the patientsubject generated by the enzymatic activity in the sample; and(iii) comparing the PTM state data set of (ii) with a PTM standard data set that includes PTM state data diagnostic for the disease or medical condition, thereby determining the PTM state of the biological sample from athe subject suspected of having said disease or medical conditionin the subject.

2. The methodassay of claim 1, wherein the PTM standard data set is generated from PTM data from one or more individuals known to have the disease or medical condition and one or more individuals who do not have the disease or medical condition.

3. The methodassay of claim 1, further comprising performing step (i) in the presence and absence of a drug, and comparing the pattern of protein PTM obtained under the effectspresence of athe drug to the pattern of PTM obtained in the absence of the drug.

4. The methodassay of claim 3, wherein the changes in the pattern of protein PTM under the effectspresence of a drug are obtained by comparing samples obtained from the subject before and after administration of the drug.

5. The methodassay of claim 1, wherein the functional biological sample is a bodily fluid selected from the group consisting of serum, plasma, and cerebrospinal fluid.

6. The methodassay of claim 1, wherein the functional biological sample isis a cell extract obtained from a frozen or cryopreserved biological sample.

7. The methodassay of claim 1, wherein the array comprising a plurality of proteins, comprises at least one protein, protein fragment or peptide attached to the array without an added tag.

8. The methodassay of claim 1, wherein the array comprising a plurality of proteins comprises at least one protein, protein fragment or peptide attached to the array with a C-terminal or N-terminal tag.

9. The methodassay of claim 1, wherein a plurality of PTM or PTM alterations thereof are identified simultaneously.

10. The methodassay of claim 1, wherein the solid state array is selected from the group consisting of protein arrays on microchips, ELISA plates with immobilized proteins attached on the plates, protein-coated beads, and microfluidic chips coated with desired proteins.

11. The methodassay of claim 1, wherein the identifying is performed using an antibody or antigen-binding fragment thereof, a natural or recombinant ligand, a small molecule, a modifying moiety, or a biochemical analysis capable of detecting the PTM or PTM alteration.

12. The methodassay of claim 11, wherein the comparison produces a pattern of protein PTM that is diagnostic for a disease or medical condition.

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