US20100160177A1

US20100160177A1 - Diagnostic method based on large scale identification of post-translational modification of proteins

Info

Publication number: US20100160177A1
Application number: US12/696,866
Authority: US
Inventors: Yifat Merbl; Marc W. Kirschner
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 2008-10-17
Filing date: 2010-01-29
Publication date: 2010-06-24
Also published as: US20220252612A1; US11333668B2; WO2010044892A1; US20150309044A1

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

This application 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

This application was made with Government Support under grant No. GM039023 awarded by the National Institutes of Health. The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

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.
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

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.
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.

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.

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.
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.
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.
In another embodiment of this aspect, the functional cell extract is obtained from a frozen or cryopreserved sample.
In another embodiment of this aspect, an additional cellular energy source in the form of ATP is provided to the functional cell extract.
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.
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.
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.
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.
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.
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.
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.
In another embodiment of this aspect, 2-10 PTM or PTM alterations thereof are identified simultaneously.
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.
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.
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.
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.
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.
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

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.

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.

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.

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).

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 emit. 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 Emil; 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.

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

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

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

FIG. 8 shows the signal—background values for the buffer spots on five representative protein microarrays.

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

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.

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

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

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.

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.

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.

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

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.
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.
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.
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.

(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.

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.
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.
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.
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.
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.
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.
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), Lis samine 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.
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.
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, deufmylation, 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.
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, σ-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.
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.
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.
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 El enzyme. The amino acid sequence of human ubiquitin is:

(SEQ ID NO: 1)

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQL

EDGRTLSDYNIQKESTLHLVLRLRGG.

As used herein, removal of one or more ubiquitin molecules is known as “deubiquitination.”
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
As used herein, “ufmylation” refers to the process whereby the ubiquitin-like modifier Ufm-1 is covalently attached to a protein. As used herein, “deufmylation” refers to the process whereby the ubiquitin-like modifier Ufm-1 is removed from a protein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.

TABLE 1

	Ubiquitin
Ubiquitin-Like	Sequence	E1-E2-E3 Conjugating
Modifier	Homology (%)	Enzymes Deconjugating Enzyme (DCE)	Substrates	Functions

ISG15 (UCRP)	29, 27	E1: UBE1L; E2: UBCH8	PLCγ1, JAK1, STAT1,	Positive regulator of IFN-related
(2 ubiquitins)			ERK1/2, serpin 2a	immune response, potentially
				involved in cell growth and
				differentiation
FUB1 (MNSFβ)	37	NA	TCR-α-like protein, Bcl-G	Negative regulator of leukocyte
				activation and proliferation
NEDD8 (Rub1)	58	E1: APPBP1-UBA3; E2: UBC12; E3:	cullins, p53, Mdm2, synphilin-1	Positive regulator of ubiquitin E3s;
		Roc1, Mdm2; DCE: DEN1/NEDP1,		directs to proteasomal degradation
		UCH-L1, UCH-L3, USP21, COP9
FAT10 (2	29, 36	NA	MAD2	Cell cycle checkpoint for spindle
ubiquitins)				assembly, directs to proteasomal
				degradation
SUMO-1 (SMT3C,	18	E1: SAE-1/-2 (AOS1-UBA2); E2:	Glut1, Glut4, c-Jun, lκBα, p53,	Control of protein stability, function,
GMP1, UBL1)		UBC9; E3: RanBP2, Pc2, PIAS	Mdm2, SOD-1, RXRα, NEMO,	and localization, antagonist to
		superfamily; DCE: SENP-1 and -2	PML, Sam68, RanGAP1,	ubiquitin, overlap with SUMO-2/-3
		(Ulp-1 and -2), SUSP4	RanBP2, ADAR1, PCNA,
			Drp1, STAT-1, Sp3, thymine-
			DNA glycosylase,
			topoisomerase II
SUMO-2 (SMT3B);	16	E1: SAE-1/-2; E2: UBC9; DCE:	RanGAP1, C/EBPβ1,	Transcription regulation, cell cycle
SUMO-3 (SMT3A)		SENP-3 and -5	topoisomerase II, thymine-	progression
			DNA glycosylase
Apg 8	10	E1: Apg7; E2: Apg3; DCE: Apg4	Phosphatidylethanolamine	Autophagy, cytoplasm-to-vacuole
				targeting
Apg 12	17	E1: Apg7; E2: Apg10	Apg 5	Autophagy, cytoplasm-to-vacuole
				targeting
Urm1	12	E1: Uba4	Ahp1	Potential role in oxidative stress
				response
UBL5 (Hub1)	25	NA	CLK4, Snu66, Sph1, Hbt1	Pre-mRNA splicing, appetite
				regulation
Ufm1	16	E1: Uba5; E2: Ufc1	NA	Potential role in endoplasmic stress
				response

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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.

TABLE 2

PTM Detected/Antibody	Catalog Number	Company

Ubiquitin monoclonal mouse monoclonal	AB-001	Cell
		Signaling
SUMO2 polyclonal mouse polyclonal	AB-S80	Cell
		Signaling
SUMO2 monoclonal mouse monoclonal	AB-S81	Cell
		Signaling
SUMO3 MaxPab polyclonal mouse polyclonal	AB-S90	Cell
		Signaling
SUMO3 polyclonal mouse polyclonal	AB-S91	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S92	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S93	Cell
		Signaling
SUMO4 MaxPab polyclonal mouse polyclonal	AB-S95	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S96	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S97	Cell
		Signaling
Anti-NEDD8 rabbit polyclonal	A-812	Cell
		Signaling
Anti-UBE1L (E1) rabbit polyclonal	A-306	Cell
		Signaling
Anti-ISG15 rabbit polyclonal	A-600	Cell
		Signaling
UBE2L6 (UbcH8) MaxPab polyclonal mouse	AB-242	Cell
polyclonal		Signaling
UBE2L6 polyclonal mouse polyclonal	AB-243	Cell
		Signaling
UBE2L6 (UbcH8) monoclonal mouse monoclonal	AB-244	Cell
Ubiquitin monoclonal mouse monoclonal	AB-001	Cell
		Signaling
SUMO2 polyclonal mouse polyclonal	AB-S80	Cell
		Signaling
SUMO2 monoclonal mouse monoclonal	AB-S81	Cell
		Signaling
SUMO3 MaxPab polyclonal mouse polyclonal	AB-S90	Cell
		Signaling
SUMO3 polyclonal mouse polyclonal	AB-S91	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S92	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S93	Cell
		Signaling
SUMO4 MaxPab polyclonal mouse polyclonal	AB-S95	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S96	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S97	Cell
		Signaling
Anti-NEDD8 rabbit polyclonal	A-812	Cell
		Signaling
Anti-UBE1L (E1) rabbit polyclonal	A-306	Cell
		Signaling
Anti-ISG15 rabbit polyclonal	A-600	Cell
		Signaling
UBE2L6 (UbcH8) MaxPab polyclonal mouse polyclonal	AB-242	Cell
		Signaling
UBE2L6 polyclonal mouse polyclonal	AB-243	Cell
		Signaling
		Signaling
ISG15 MaxPab polyclonal rabbit polyclonal	AB-I10	Cell
		Signaling
ISG15 monoclonal clonal mouse monoclonal	AB-I11	Cell
		Signaling
Anti-UFM1 rabbit polyclonal	A-500	Cell
		Signaling
APG3 polyclonal mouse recombinant	AB-A10APG3	Cell
		Signaling
APG3 monoclonal mouse monoclonal	AB-A11APG3	Cell
		Signaling
APG4B polyclonal rabbit polyclonal	AB-A20APG4B	Cell
		Signaling
APG4C MaxPab polyclonal mouse polyclonal	AB-A21APG4C	Cell
Ubiquitin monoclonal mouse monoclonal	AB-001	Cell
		Signaling
SUMO2 polyclonal mouse polyclonal	AB-S80	Cell
		Signaling
SUMO2 monoclonal mouse monoclonal	AB-S81	Cell
		Signaling
SUMO3 MaxPab polyclonal mouse polyclonal	AB-S90	Cell
		Signaling
SUMO3 polyclonal mouse polyclonal	AB-S91	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S92	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S93	Cell
		Signaling
SUMO4 MaxPab polyclonal mouse polyclonal	AB-S95	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S96	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S97	Cell
		Signaling
Anti-NEDD8 rabbit polyclonal	A-812	Cell
		Signaling
Anti-UBE1L (E1) rabbit polyclonal	A-306	Cell
		Signaling
Anti-ISG15 rabbit polyclonal	A-600	Cell
		Signaling
UBE2L6 (UbcH8) MaxPab polyclonal mouse	AB-242	Cell
polyclonal		Signaling
UBE2L6 polyclonal mouse polyclonal	AB-243	Cell
		Signaling
		Signaling
APG4C polyclonal rabbit polyclonal	AB-A22APG4C	Cell
		Signaling
APG5 monoclonal mouse monoclonal	AB-A25APG5	Cell
		Signaling
APG7 MaxPab polyclonal mouse polyclonal	AB-A30APG7	Cell
		Signaling
APG7 polyclonal rabbit polyclonal	AB-A31APG7	Cell
		Signaling
APG9A polyclonal rabbit polyclonal	AB-A40APG9	Cell
		Signaling
APG10 polyclonal rabbit polyclonal	AB-A50APG10	Cell
		Signaling
APG10 polyclonal rabbit polyclonal	AB-A51APG10	Cell
Ubiquitin monoclonal mouse monoclonal	AB-001	Cell
		Signaling
SUMO2 polyclonal mouse polyclonal	AB-S80	Cell
		Signaling
SUMO2 monoclonal mouse monoclonal	AB-S81	Cell
		Signaling
SUMO3 MaxPab polyclonal mouse polyclonal	AB-S90	Cell
		Signaling
SUMO3 polyclonal mouse polyclonal	AB-S91	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S92	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S93	Cell
		Signaling
SUMO4 MaxPab polyclonal mouse polyclonal	AB-S95	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S96	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S97	Cell
		Signaling
Anti-NEDD8 rabbit polyclonal	A-812	Cell
		Signaling
Anti-UBE1L (E1) rabbit polyclonal	A-306	Cell
		Signaling
Anti-ISG15 rabbit polyclonal	A-600	Cell
		Signaling
UBE2L6 (UbcH8) MaxPab polyclonal mouse	AB-242	Cell
polyclonal		Signaling
UBE2L6 polyclonal mouse polyclonal	AB-243	Cell
		Signaling
		Signaling
APG12 MaxPab polyclonal mouse polyclonal	AB-A64APG12	Cell
		Signaling
APG12 polyclonal rabbit polyclonal	AB-A65APG12	Cell
		Signaling
APG12 monoclonal mouse monoclonal	AB-A66APG12	Cell
		Signaling
URM1 polyclonal rabbit polyclonal	AB-O30	Cell
		Signaling
Anti Fat10 (Protein derived)	PW9680-002	Biomol
anti-Fat10 Polyclonal	PW9585-0025	Biomol
anti-URM1 polyclonal	PW9595-0025	Biomol
anti-FUB1 polyclonal	PW9615-0025	Biomol
Mouse Anti-O-GlcNAc Monoclonal Antibody	sc-81483	Santa Cruz
Ubiquitin monoclonal mouse monoclonal	AB-001	Cell
		Signaling
SUMO2 polyclonal mouse polyclonal	AB-S80	Cell
		Signaling
SUMO2 monoclonal mouse monoclonal	AB-S81	Cell
		Signaling
SUMO3 MaxPab polyclonal mouse polyclonal	AB-S90	Cell
		Signaling
SUMO3 polyclonal mouse polyclonal	AB-S91	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S92	Cell
		Signaling
SUMO3 monoclonal mouse monoclonal	AB-S93	Cell
		Signaling
SUMO4 MaxPab polyclonal mouse polyclonal	AB-S95	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S96	Cell
		Signaling
SUMO4 polyclonal rabbit polyclonal	AB-S97	Cell
		Signaling
Anti-NEDD8 rabbit polyclonal	A-812	Cell
		Signaling
Anti-UBE1L (E1) rabbit polyclonal	A-306	Cell
		Signaling
Anti-ISG15 rabbit polyclonal	A-600	Cell
		Signaling
UBE2L6 (UbcH8) MaxPab polyclonal mouse	AB-242	Cell
polyclonal		Signaling
UBE2L6 polyclonal mouse polyclonal	AB-243	Cell
		Signaling
S-nitrosocysteine antibody	ab50185	Abcam
Acetylated-Lysine Antibody	#9441	Cell
		Signaling
acetyl Lysine antibody	ab76	Abcam
Citrulline polyclonal antibody	PAB0068	Abnova

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.
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.
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.
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.
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.
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).
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.
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.
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.
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%.
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).
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.
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.
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.
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.

TABLE 3

		MODIFIED
PTM	DISEASE	PROTEIN	REFERENCE	TITLE

Ubiquitination	Cancer/tumor	cMyc, HectH9 (E3	33	The ubiquitin ligase
		ligase)		HectH9 regulates
				transcriptional activation
				by Myc and is essential for
				tumor cell proliferation
Ubiquitination	Cancer/tumor, Breast	BRCA1 (E3 ligase)	34	Ubiquitination and
	and ovarian cancer			proteasomal degradation of
				the BRCA1 tumor
				suppressor is regulated
				during cell cycle
				progression.
SUMOylation	Cancer/tumor	Ubc9 (E2 conjugating	35	A role for Ubc9 in
		enzyme)		tumorigenesis
Ubiquitination	Alzheimers disease	Bcl-2	36	Inhibition of the ubiquitin-
				proteasome system in
				Alzheimer's Disease
Glycosylation	Alzheimers disease	tau		37	Glycosylation of
				microtubule-associated
				protein tau: an abnormal
				posttranslational
				modification in
				Alzheimer's disease
K48-linked and K63-linked	Parkinson's disease	synphilin-1, parkin, α-	38	Parkin mediated lysine 63-
ubiquitination		synuclein, UCHL1		linked polyubiquitination: a
				link to protein inclusions
				formation in Parkinson's
				and other conformational
				diseases?
Ubiquitination	Parkinson's Disease,	Parkin	39	Parkin-mediated K63-
	Autophagy			linked polyubiquitination: a
				signal for targeting
				misfolded proteins to the
				aggresome-autophagy
				pathway.
Ubiquitination	Neurodegenerative	P62		40	Lysine 63-linked
	Diseases			polyubiquitin potentially
				partners with p62 to
				promote the clearance of
				protein inclusions by
				autophagy.
Acetylation, deacetylation,	Neurologic and	HDAC	41	Epigenetic targets of
methylation	psychiatric disorders			HDAC inhibition in
	including Huntington's			neurodegenerative and
	disease, Parkinson's			psychiatric disorders.
	disease, anxiety and
	mood disorders,
	Rubinstein-Taybi
	syndrome, and Rett
	syndrome
Nedd8ylation	Neurodegenerative	NEDD8	42	Accumulation of NEDD8
	Diseases, Parkinson's			in neuronal and glial
	disease and Rosenthal			inclusions of
	fibres in astrocytoma			neurodegenerative
				disorders.
	Neurodegenerative	Mad2, BubR1	43	Inhibitory factors
	diseases	associated to cDc20		associated with anaphase-
				promoting
				complex/cylosome in
				mitotic checkpoint.
Ubiquitination	Cell Cycle progression		44	Ubiquitin dependence of
				selective protein
				degradation demonstrated
				in the mammalian cell
				cycle mutant ts85.
	Cell Cycle progression	cyclin	45	Cyclin: a protein specified
				by maternal mRNA in sea
				urchin eggs that is
				destroyed at each cleavage
				division.
	Cell Cycle progression	APC/C (cDc20, CDH1	46	Control of mitotic
		and MAD2)		transitions by the anaphase-
				promoting complex.
Conjugation	Cell Cycle progression	cyclin	47	Cyclin is degraded by the
				ubiquitin pathway.
Ubiquitination	Cell Cycle progression	Cdc34, CDK activity-	48	How proteolysis drives the
		by degrading CDK		cell cycle
		activators or inhibitors
Ubiquitination	Cell Cycle progression	APC/C (cDc20,	49	Ubiquitination by the
		MAD2)		anaphase-promoting
				complex drives spindle
				checkpoint inactivation.
Ubiquitination, phosphorylation,	DNA damage and repair	ATR/MRN complex	50	Twists and turns in the
methylation DNA damage and				function of DNA damage
repair				signaling and 5 repair
				proteins by PTMs.
Acetylation, methylation,	Huntington disease	Histone (H2A, H2B,	51	Mechanisms of disease:
phosphorylation, ubiquitination		H3 and H4)		Histone modifications in
and SUMOylation				Huntington's disease.
SUMOylation	Huntington disease	Huntingtin (Httex1p)	52	SUMO modification of
				Huntingtin and
				Huntington's disease
				pathology.
Ubiquitination, SUMOylation,	NFkB pathway	IkappaB kinase (IKK)	53	PTMs regulating the
phosphorylation, acetylation and		complex, the IkappaB		activity and function of the
nitrosylation		proteins and the NF-		nuclear factor kappa B
		kappaB		pathway.
SUMOylation	Neuronal Intranuclear	SUMOylation	54	SUMOylation substrates in
	Inclusion disease (NIID)	substrates:		neuronal intranuclear
		Promyelocytic		inclusion disease.
		leukaemia protein
		(PML) and
		RanGAP1.HDAC4
SUMOylation	Type
1 diabetes	M55V substitution of	55	SUMO wrestling with type
		SUMO4
		1 diabetes.
SUMOylation	Polyglutamine Diseases	ESCA1 and ESCA2	56	Enhanced SUMOylation in
				polyglutamine diseases
Ubiquitination	Kidney cancers	HIF-alpha	57	The role of von Hippel-
				Lindau tumor suppressor
				protein and hypoxia in
				renal clear cell carcinoma.
Neddylation, SUMOylation,	Renal cell carcinomas,	pVHL, NEDD8	58	The von Hippel-Lindau
	pheochromocytomas,	conjugation to Cul-2		tumor suppressor gene
	and vascular tumors of			product promotes, but is
	the central nervous			not essential for, NEDD8
	system			conjugation to cullin-5 2.
SUMOylation	Diabetes mellitus,	ERK5, Ubc9 (SUMO	59	Effects of MEK5/ERK5
	diabetic ventricular	E2 conjugase) or		association on small
	dysfunction	PIAS1 (E3 ligase)		ubiquitin-related
				modification of ERK5:
				implications for diabetic
				ventricular dysfunction
				after myocardial infarction.
Ubiquitination, SUMOylation	Parkinson's,	αSYN (PARK1), UCH-	60	The ubiquitin proteasome
	Alzheimer's,	L1, DJ-1 binds to the		system in
	Huntington's, Prion and	SUMO E3 PIASx, Aβ		neurodegenerative
	amyotrophic lateral	and tau, UBB + 1etc . . .		diseases: sometimes the
	sclerosis			chicken, sometimes the
				egg.
Methylation, deimination, and	Multiple Sclerosis	MBP	61	Multiple sclerosis: an
phosphorylation				important role for PTMs of
				myelin basic protein in
				pathogenesis.
Glycosylation	Autoimmunity,	IgG and IgA1	62	Plasma proteins
	Rheumatoid arthritis and			glycosylation and its
	IgA nephropathy			alteration in disease.
SUMOylation	Parkinson	DJ-1	63	Proper SUMO-1
				conjugation is essential to
SUMOylation	Parkinson	DJ-1, and pyrimidine	64	DJ-1 to exert its full
		tract-binding protein-		activities.
		associated splicing		DJ-1 transcriptionally up-
		factor (PSF)		regulates the human
				tyrosine hydroxylase by
				inhibiting the sumoylation
				of pyrimidine tract-binding
				protein-associated splicing
				factor.
Ubiquitination, phosphorylation	Cancer	p53	65	Ubiquitination,
and acetylation				phosphorylation and
				acetylation: the molecular
				basis for p53 regulation.
Phosphorylation	Cancer	Fra-1	66	Accumulation of Fra-1 in
				ras-transformed cells
				depends on both
				transcriptional
				autoregulation and MEK-
				dependent posttranslational
				stabilization.
Phophorylation	Cancer	NF-kappa B	67	Inhibition of constitutive
				NF-kappa B activity by I
				kappa B alpha M
				suppresses tumorigenesis.
Ubiquitination, SUMOylation	Cancer	Smad4	68	Sumoylation of Smad4, the
				common Smad mediator of
				transforming growth
				factor-beta family
				signaling.
Phophorylation	Uterine leiomyomas	Ref-1	69	Altered PTM of redox
				factor 1protein in human
				uterine smooth muscle
				tumors.
Phophorylation	tumorigenesis,	p53, GSK3beta	70	Glycogen synthase kinase3
	differentiation and			beta phosphorylates serine
	apoptosis			33 of p53 and activates
				p53's transcriptional
				activity.
Phophorylation	tumorigenesis	pp60c-src	71	pp60c-src in human
				melanocytes and melanoma
				30 cells exhibits elevated
				specific activity and
				reduced tyrosine 530
				phosphorylation compared
				to human fibroblast pp60c-
				src.
Phophorylation	tumorigenesis	P120	72	Abelson murine leukemia
				virus
				transformationdefective
				mutants with impaired
				P120 associated protein
				kinase activity.
Glycosylation	Prion Disease	PrP	73	Asparagine-linked
				glycosylation of the scrapie
				and cellular prion proteins.
Ubiquitination	Fanconi anemia	FANCD2	74	Fanconi anemia: causes
				and consequences of
				genetic instability.
Ubiquitination	Fanconi anemia	FANCD2, catalytic	75	A novel ubiquitin ligase is
		subunit PHF9(FANCL)		deficient in Fanconi
				anemia.
Ubiquitination	Aging	BRCA1; PCNA;	76	Aging and the
		NFκB; p27;		ubiquitinome: traditional
		SNEV^Prp19/Pso4		and non-traditional
				functions of ubiquitin in
				aging cells and tissues.
Ubiquitination, SUMOylation,	Aging		77	Aging and dietary
Oxydation				restriction effects on
				ubiquitination,
				sumoylation, and the
				proteasome in the heart.
Ubiquitination	Aging	DAF-16, RLE-1 (E3	78	RLE-1, an E3 ubiquitin
		ligase)		ligase, regulates C. elegans
				aging by catalyzing DAF-
				16 polyubiquitination.
SUMOylation	Aging	POMP-1	79	Effects of aging and dietary
				restriction on
				ubiquitination,
				sumoylation, and the
				proteasome in the spleen.
	Aging	Decrease of expressed	80	Caretaker or undertaker?
		Proteasome		The role of the proteasome
		proteins: S9:Rpn6		in aging
		(p44.5), Rpn5 (p55), a2
		(HC3), a7(HC8),
		S7:Rpt1 (MSS1) and
		S10b:Rpt4 (p42)
S-nitrosylation, Ubiquitination	Parkinson's disease	parkin	81	Nitrosative stress linked to
				sporadic Parkinson's
				disease: S-nitrosylation of
				parkin regulates its E3
				ubiquitin ligase activity.
Glycosylation	Virus related diseases	penv9, penv14	82	Glycosylation inhibitors
				block the expression of
				LAV/HTLV-III (HIV)
				glycoproteins.
Glycosylation	Virus related diseases	gp46	83	Immunogenicity and
				conformational properties
				of an N-linked glycosylated
				peptide epitope of human
				T-lymphotropic virus type
				1 (HTLVI).
Glycosylation	Virus related diseases	peroxiredoxin	1 and	84	Posttranslational
		HTLV-1-p24-(gag)		glycosylation of target
				proteins implicate
				molecular mimicry in the
				pathogenesis of HTLV-1
				associated neurological
				disease.
Glycosylation	Virus related diseases	gp	100	85	A glycopolypeptide (gp
				100) is the main antigen
				detected by HTLV-III
				antisera.
Citrullination/deimination	Multiple Sclerosis,	Myelin basic protein	86	A tale of two citrullines-
	Diabetes, Alzheimer's	(MBP)		structural and functional
				aspects of myelin basic
				protein deimination in
				health and 5 disease.
OGlcNAc	Cardiac dysfunction	SP1, eNOS,	87	O-GlcNAc modification of
				nucleocytoplasmic proteins
				and diabetes.
OGlcNAc	Diabetes, Alzheimer's	tau, β-amyloid	88	O-GlcNAc modification in
	disease	precurssor, AP-3,		diabetes and Alzheimer's
		synapsin-I,		disease.
		Neurofilament H, L, M.
		IRS, GS, PDX-1,
		eNOS, SP1
OGlcNAc	Diabetes		89	A bittersweet modification:
				O-GlcNAc and cardiac
				dysfunction.
OGlcNAc	Diabetes	Sp1(but also	90	PTM by O15 GlcNAc:
		metionned the serum		another way to change
		response factor, c-myc,		protein function.
		estrogen receptors and
		RNA pol II)
Various PTMs	Atherosclerosis; celiac	αB-crystallin, MBP,	91	Posttranslational protein
	disease; autoimmune	Fibrin, Type II		modifications: new flavors
	encephalomyelitis;	collagen, MBP Ac1-1,		in the menu of
	multiple sclerosis;	Sm D1, D3, Wheat		autoantigens.
	systemic lupus	gliadin, LDL, SnRNPD
	erythematosus; collagen-
	induced arthritis;
	rheumatoid arthritis
Various PTMs	Multiple sclerosis/EAE,	Fillagrin, Vimentin,	92	Posttranslational
	Collagen-induced	H2B		modifications of self-
	arthritis, Rheumatoid			antigens.
	arthritis, systemic lupus
	erythematosus.
Various PTMs	Rheumatoid arthritis;	trichohyalin, filaggrin	93	Modifications of arginines
	Multiple sclerosis;	and keratin, myelin		and their role in
	Systemic lupus	basic protein(MBP),		autoimmunity.
	erythematosus	fibrin, vimentin and
		nucleophosmin/B23,
		histones, Sm-D1, Sm-
		D3, Sm-ByB9, LSm4
Citrullination	Rheumatoid arthritis	Fibrin	94	Autoantigenic
				posttranslational
				modifications of proteins:
				does it apply to rheumatoid
				arthritis?

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.
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.
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.
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.
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.
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.
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.
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.
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.
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 I

Protein Ubiquitination Patterns Upon Escape from the Spindle Assembly Checkpoint in Mammalian Cells

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.
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).
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

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

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), 150mM creatine phosphate, 20 mM ATP (pH 7.4), 2 mM EGTA (pH 7.7), 20 mM MgCl₂).
Incubation of Extracts with Microarrays
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 Emil (1 mg/ml) or H₂O for 30 minutes. 100 μl of CP or G1 extracts (˜25 mg/ml) 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

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

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

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

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 (Mbl), calmodulin (Upstate), and p27 (Upstate)) were determined in the extracts by Western blotting at the indicated times.

Results

The E2-conjugating enzyme, UbcH10, has been shown to overcome the metaphase-anaphase transition (16). After arresting cells in nocodazole, concentrated extracts (apprx 25mg/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).
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, emit. 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 Emil (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.
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).
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.
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 p27) 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 emil (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 emil 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 emil 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 emil.

TABLE 4

Protein Name	Accession	p-value

histone UNFRAC. WHOLE HISTONE - known Autoantigen		0.0002
ring finger protein 128 (RNF128) transcript variant 1	NM_194463.1	0.0004
erythrocyte membrane protein band 4.1 like 5	BC054508.1	0.0004
BC013173 Homo sapiens, clone MGC: 17340	BC013173.1	0.0004
Clmodulin 2	NM_001743	0.0005
HTGN29 protein (HTGN29)	NM_020199.1	0.0006
ankyrin repeat domain 13	BC032833.2	0.0006
ribosomal protein S6 kinase 90 kDa polypeptide 4 (RPS6KA4) transcript variant 2	NM_001006944.1	0.0007
macrophage stimulating 1 receptor (c-met-related tyrosine kinase) (MST1R)	NM_002447.1	0.0008
hypothetical protein FLJ11184	BC011842.2	0.0008
PCTAIRE protein kinase 2	BC033005.1	0.0008
aurora kinase A (AURKA) transcript variant 2	NM_003600.2	0.0009
dolichyl-phosphate mannosyltransferase polypeptide 2 regulatory subunit (DPM2)	NM_152690.1	0.0009
transcript variant 2
ems1 sequence (mammary tumor and squamous cell carcinoma-associated (p80/85 src	NM_138565.1	0.0009
substrate) (EMS1)
cytochrome P450 family 26 subfamily A polypeptide 1 (CYP26A1) transcript variant 2	NM_057157.1	0.0010
KIAA0157 protein (KIAA0157)	NM_032182.2	0.0010
solute carrier family 23 (nucleobase transporters) member 2	BC013112.2	0.0011
ring finger protein 111	BC060862.1	0.0011
additional sex combs like 1 (Drosophila)	BC064984.1	0.0012
cDNA clone MGC: 39273 IMAGE: 5440834	BC024289.1	0.0012
PAS domain containing serine/threonine kinase (PASK)	NM_015148.1	0.0013
YY1 transcription factor (YY1)	NM_003403.3	0.0013
proteasome (prosome macropain) 26S subunit non-ATPase 4 (PSMD4) transcript variant 1	NM_002810.1	0.0014
hypothetical protein LOC143458 (LOC143458)	NM_174902.2	0.0014
selectin ligand interactor cytoplasmic-1 (SLIC1) transcript variant 1	NM_153337.1	0.0015
MAX interacting protein 1 (MXI1) transcript variant 2	NM_130439.1	0.0015
neural precursor cell expressed developmentally down-regulated 8 (NEDD8)	NM_006156.1	0.0016
aurora kinase B (AURKB)	NM_004217.2	0.0016
src homology three (SH3) and cysteine rich domain	BC020221.1	0.0016
hypothetical protein DKFZp762O076 (DKFZp762O076)	NM_018710.1	0.0016
Nedd4 family interacting protein 1 (NDFIP1)	NM_030571.2	0.0016
hypothetical protein FLJ36175	BC029520.1	0.0017
EGF-like repeats and discoidin I-like domains 3	BC053656.1	0.0018
hypothetical protein MGC4618 (MGC4618)	NM_032326.1	0.0019
zeta-chain (TCR) associated protein kinase 70 kDa (ZAP70) transcript variant 1	NM_001079.3	0.0019
ribosomal protein L30 (RPL30)	NM_000989.2	0.0019
feline sarcoma oncogene (FES)	NM_002005.2	0.0019
met proto-oncogene (hepatocyte growth factor receptor) (MET)	NM_000245.2	0.0021
ADP-ribosylation factor-like 7 (ARL7)	NM_005737.3	0.0022
Histone_F2a2 H2a(f2a2) - known Autoantigen		0.0022
likely ortholog of mouse gene trap locus 3 (GTL3)	NM_013242.1	0.0022
immediate early response 3 (IER3) transcript variant short	NM_003897.2	0.0023
potassium voltage-gated channel shaker-related subfamily beta member 2 (KCNAB2)	NM_003636.1	0.0023
immunoglobulin heavy constant gamma 1 (G1m marker)	BC014667.1	0.0024
ring finger protein 4 (RNF4)	NM_002938.2	0.0025
proteasome (prosome macropain) 26S subunit non-ATPase 4 (PSMD4) transcript variant 2	NM_153822.1	0.0026
chromosome 6 open reading frame 145 (C6orf145)	NM_183373.2	0.0027
neurotrophic tyrosine kinase receptor type 1 (NTRK1) transcript variant 3	NM_001007792.1	0.0028
pleckstrin homology domain containing family G member 5 (PLEKHG5) transcript	NM_020631.2	0.0028
variant 1
Sjogren syndrome antigen A1 (52 kDa ribonucleoprotein autoantigen SS-A/Ro) (SSA1)	NM_003141.2	0.0028
interferon stimulated gene 20 kDa (ISG20)	NM_002201.3	0.0028
WD repeat domain 45 (WDR45) transcript variant 1	NM_007075.3	0.0029
TANK-binding kinase 1 (TBK1)	NM_013254.2	0.0029
chromosome 16 open reading frame 5	BC002882.1	0.0030
insulin-like growth factor 1 receptor (IGF1R)	NM_000875.2	0.0030
ring finger protein 111	BC010369.1	0.0031
G protein-coupled receptor kinase 4 (GRK4) transcript variant 2	NM_001004056.1	0.0032
v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN)	NM_002350.1	0.0033
RAS-like family 10 member B	BC041133.1	0.0034
hypothetical protein MGC11257 (MGC11257)	NM_032350.3	0.0035
chromosome 7 open reading frame 2 (C7orf2)	NM_022458.2	0.0035
expressed in T-cells and eosinophils in atopic dermatitis (ETEA)	NM_014613.1	0.0036
mitogen-activated protein kinase kinase kinase 11 (MAP3K11)	NM_002419.2	0.0036
casein kinase 1 alpha 1 (CSNK1A1) transcript variant 1	NM_001025105.1	0.0038
zeta-chain (TCR) associated protein kinase 70 kDa transcript variant 1	BC053878.1	0.0038
hypothetical gene LOC128439 (LOC128439)	NM_139016.2	0.0038
hypothetical protein MGC17403 (MGC17403)	NM_152634.1	0.0039
N-glycanase 1 (NGLY1)	NM_018297.2	0.0039
signal recognition particle 19 kDa	BC010947.1	0.0040
DNA fragmentation factor 40 kDa beta polypeptide (caspase-activated DNase) (DFFB)	NM_001004285.1	0.0040
transcript variant 3
casein kinase 1 delta (CSNK1D) transcript variant 1 Not full-length.	NM_001893.3	0.0042
dendritic cell-derived ubiquitin-like protein (DC-UbP)	NM_152277.1	0.0043
cDNA clone MGC: 3432 IMAGE: 2959461	BC013957.1	0.0043
DnaJ (Hsp40) homolog subfamily B member 12 (DNAJB12) transcript variant 1	NM_001002762.1	0.0043
solute carrier family 36 (proton/amino acid symporter) member 4	BC047374.1	0.0044
SMT3 suppressor of mif two 3 homolog 1 (yeast) (SUMO1) transcript variant 1	NM_003352.4	0.0044
similar to hypothetical protein FLJ25555	BC044239.1	0.0049
lysosomal-associated protein transmembrane 4 alpha (LAPTM4A)	NM_014713.2	0.0050
KIAA1458 protein	BC031691.2	0.0051
interleukin 17E (IL17E) transcript variant 1	NM_022789.2	0.0053
serum/glucocorticoid regulated kinase (SGK)	NM_005627.1	0.0053
hypothetical protein FLJ10156	BC005004.1	0.0054
thousand and one amino acid protein kinase (TAO1)	NM_004783.1	0.0054
ADP-ribosylation-like factor 6 interacting protein 4 (ARL6IP4)	NM_016638.1	0.0054
zinc finger protein 313 (ZNF313)	NM_018683.2	0.0055
solute carrier family 6 (neurotransmitter transporter) member 15	BC022253.1	0.0055
XM_378350.2	XM_378350.2	0.0057
low density lipoprotein receptor-related protein 10 (LRP10)	NM_014045.1	0.0060
arrestin domain containing 3 (ARRDC3)	NM_020801.1	0.0062
cyclin-dependent kinase inhibitor 1B (p27 Kip1) (CDKN1B)	NM_004064.2	0.0062
p53-regulated DDA3 (DDA3)	NM_032636.2	0.0065
calcium/calmodulin-dependent protein kinase IV (CAMK4)	NM_001744.2	0.0066
BC015569 Homo sapiens, Similar to SRp25 nuclear protein	BC015569.1	0.0066
chromosome 6 open reading frame 201 (C6orf201)	NM_206834.1	0.0067
tripartite motif-containing 52 (TRIM52)	NM_032765.1	0.0067
hypothetical protein FLJ38628 (FLJ38628)	NM_152267.2	0.0071
vasopressin-induced transcript	BC000877.1	0.0074
Ro-52 Ro-52 - known Autoantigen		0.0074
cyclin G2	BC032518.1	0.0076
mitogen-activated protein kinase kinase 6 (MAP2K6) transcript variant 2; mutant	NM_031988.1	0.0077
protein: MAP2K6 mutant
conserved helix-loop-helix ubiquitous kinase (CHUK)	NM_001278.3	0.0078
aurora kinase C (AURKC) transcript variant 1	NM_001015878.1	0.0079
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 (DYRK3) transcript	NM_001004023.1	0.0080
variant 2
cullin 3 (CUL3)	NM_003590.2	0.0080
hepatocyte growth factor-regulated tyrosine kinase substrate (HGS)	NM_004712.3	0.0084
B lymphoid tyrosine kinase (BLK)	NM_001715.2	0.0084
hypothetical protein MGC40579 (MGC40579)	NM_152776.1	0.0086
NIMA (never in mitosis gene a)-related kinase 9 (NEK9)	NM_033116.2	0.0086
solute carrier family 1 member 1 (SLC1A1) nuclear gene encoding mitochondrial protein	NM_004170.2	0.0086
Homo sapiens, SWI/SNF related, matrix associated, actin dependent regulator of	BC018953.1	0.0086
chromatin, subfamily d, member 2
calcium binding protein 4	BC033167.1	0.0088
chromosome 19 open reading frame 28 (C19orf28)	NM_174983.2	0.0088
ubiquitin-activating enzyme E1-like (UBE1L)	NM_003335.2	0.0090
regenerating islet-derived 1 alpha (pancreatic stone protein pancreatic thread protein)	BC005350.1	0.0090
DnaJ (Hsp40) homolog subfamily B member 6 transcript variant 2	BC000177.2	0.0091
calcium/calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B)	NM_001220.3	0.0093
transcript variant1
ubiquitin-conjugating enzyme E2-like	BC064566.1	0.0094
melanoma antigen family B 1 (MAGEB1)	NM_002363.1	0.0097
secretory carrier membrane protein 3 (SCAMP3) transcript variant 1	NM_005698.2	0.0097
hypothetical protein LOC255330	BC042038.1	0.0099

Example II

Ubiquitination of Human Brain Proteins in Alzheimer's Disease

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.
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 III

Protein Ubiquitination in Cerebrospinal Fluid (CSF) from a Patient with Brain Tumor

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.
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 IV

Analysis of Protein Ubiquitination in Normal Human Cerebrospinal Fluid (CSF) Sample

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).
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.

TABLE 5

Accession	Protein Description

NM_006259	S100 calcium binding protein A14 (S100A14), mRNA
NM_020672	Williams Beuren syndrome chromosome region 22, mRNA
	(cDNA clone MGC: 2022 IMAGE: 3544156)
BC001780	zinc finger CCCH-type containing 10 (ZC3H10), mRNA
NM_032786	chemokine (C—X—C motif) ligand 11 (CXCL11), mRNA
NM_032357	ankyrin repeat and BTB (POZ) domain containing 1
	(ABTB1), transcript variant 1, mRNA
NM_006597	interleukin
1, alpha (IL1A), mRNA
NM_032548	v-akt murine thymoma viral oncogene homolog 3 (protein
	kinase B, gamma) (AKT3), transcript variant 1, mRNA
NM_174902	serine carboxypeptidase	1, mRNA (cDNA clone IMAGE:
	4328599), partial cds
NM_000961	v-raf murine sarcoma 3611 viral oncogene homolog
	(ARAF), mRNA
NM_002609	tec protein tyrosine kinase (TEC), mRNA
NM_025160	myotilin (MYOT), transcript variant 1, mRNA
NM_017881	platelet-derived growth factor receptor, beta polypeptide
	(PDGFRB), mRNA
NM_033505	SELI selenoprotein I (SLE1)

Example V

Proteins Modified with Ubiquitin-Like Modifiers Upon Mitotic Release

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, sumol, 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.
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.

TABLE 6

GenBank
Accession	Gene Symbol	Name

Ubiquitin

BC001396	C9ORF32	CHROMOSOME 9 OPEN READING FRAME 32
BC004967	UBAC1	UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1
BC007581	ALDH4A1	ALDEHYDE DEHYDROGENASE 4 FAMILY, MEMBER
		A1
BC008720	CRELD1	DKFZP566D213 PROTEIN
BC010369	RNF111	RING FINGER PROTEIN 111
BC011399	SYK	SPLEEN TYROSINE KINASE
BC013173	RSPRY1	RING FINGER AND SPRY DOMAIN CONTAINING 1
BC015219	RBCK1	CHROMOSOME 20 OPEN READING FRAME 18
BC020221	STAC	SH3 AND CYSTEINE RICH DOMAIN
BC021988	NDFIP2	NEDD4 FAMILY INTERACTING PROTEIN 2
BC032518	CCNG2	CYCLIN G2
BC036540	LOC400120	HYPOTHETICAL LOC400120
BC041133	RASL10B	RAS-LIKE, FAMILY 10, MEMBER B
BC044239	ANKRD13D	ANKYRIN REPEAT DOMAIN 13 FAMILY, MEMBER D
BC046151	TOM1	TARGET OF MYB1 (CHICKEN)
BC048970	TTLL7	TUBULIN TYROSINE LIGASE-LIKE FAMILY,
		MEMBER 7
BC056240	SPRR1B	SMALL PROLINE-RICH PROTEIN 1B (CORNIFIN)
BC066340	BLOC1S1	BIOGENESIS OF LYSOSOME-RELATED ORGANELLES
		COMPLEX-1, SUBUNIT 1
NM_000875	IGF1R	INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR
NM_001004056	GRK4	G PROTEIN-COUPLED RECEPTOR KINASE 4
NM_001220	CAMK2B	CALCIUM/CALMODULIN-DEPENDENT PROTEIN
		KINASE (CAM KINASE) II BETA
NM_002103	GYS1	GLYCOGEN SYNTHASE 1 (MUSCLE)
NM_002378	MATK	MEGAKARYOCYTE-ASSOCIATED TYROSINE
		KINASE
NM_002648	PIM1	PIM-1 ONCOGENE
NM_002810	PSMD4	PROTEASOME (PROSOME, MACROPAIN) 26S
		SUBUNIT, NON-ATPASE, 4
NM_003045	SLC7A1	SOLUTE CARRIER FAMILY 7 (CATIONIC AMINO
		ACID TRANSPORTER, Y+ SYSTEM), MEMBER 1
NM_003403	YY1	YY1 TRANSCRIPTION FACTOR
NM_004438	EPHA4	EPH RECEPTOR A4
NM_004712	HGS	HEPATOCYTE GROWTH FACTOR-REGULATED
		TYROSINE KINASE SUBSTRATE
NM_004783	TAOK2	TAO KINASE 2
NM_005030	PLK1	POLO-LIKE KINASE 1 (DROSOPHILA)
NM_005727	TSPAN1	TETRASPANIN 1
NM_005737	ARL4C	ADP-RIBOSYLATION FACTOR-LIKE 4C
NM_006007	ZFAND5	ZINC FINGER, A20 DOMAIN CONTAINING 2
NM_006293	TYRO3	TYRO3 PROTEIN TYROSINE KINASE
NM_013242	C16ORF80	GENE TRAP LOCUS 3 (MOUSE)
NM_018215	FLJ10781	HYPOTHETICAL PROTEIN FLJ10781
NM_018384	GIMAP5	GTPASE, IMAP FAMILY MEMBER 5
NM_022905	TTC23	TETRATRICOPEPTIDE REPEAT DOMAIN 23
NM_032182	KIAA0157	KIAA0157
NM_032765	TRIM52	TRIPARTITE MOTIF-CONTAINING 52
NM_080823	SRMS	SRC-RELATED KINASE LACKING C-TERMINAL
		REGULATORY TYROSINE AND N-TERMINAL
		MYRISTYLATION SITES
NM_130439	MXI1	MAX INTERACTOR 1
NM_152285	ARRDC1	ARRESTIN DOMAIN CONTAINING 1
NM_153217	TMEM174	HYPOTHETICAL PROTEIN MGC13034
NM_153822	PSMD4	PROTEASOME (PROSOME, MACROPAIN) 26S
		SUBUNIT, NON-ATPASE, 4
NM_173541	C10ORF91	CHROMOSOME 10 OPEN READING FRAME 91
NM_194271	RNF34	RING FINGER PROTEIN 34
BC016381	NA	NA
BC004967*	UBAC1	UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1
BC010369*	RNF111	RING FINGER PROTEIN 111
BC014475*	BIRC7	LIVIN INHIBITOR-OF-APOTOSIS
BC015569*	ARL6IP4	ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTING
		PROTEIN 4
BC021988*	NDFIP2	NEDD4 FAMILY INTERACTING PROTEIN 2
BC023982*	C5ORF32	PUTATIVE NUCLEAR PROTEIN ORF1-FL49
BC025700*	AFF4	AF4/FMR2 FAMILY, MEMBER 4
BC044239*	ANKRD13D	ANKYRIN REPEAT DOMAIN 13 FAMILY, MEMBER D
BC053895*	IRS1	INSULIN RECEPTOR SUBSTRATE 1
BC054049*	ZNF364	ZINC FINGER PROTEIN 364
BC060833*	PRRG1	PROLINE RICH GLA (G-CARBOXYGLUTAMIC ACID) 1
NM_001033551*	TOM1L2	TARGET OF MYB1-LIKE 2 (CHICKEN)
NM_002019*	FLT1	FMS-RELATED TYROSINE KINASE 1 (VASCULAR
		ENDOTHELIAL GROWTH FACTOR/VASCULAR
		PERMEABILITY FACTOR RECEPTOR)
NM_002110*	HCK	HEMOPOIETIC CELL KINASE
NM_002253*	KDR	KINASE INSERT DOMAIN RECEPTOR (A TYPE III
		RECEPTOR TYROSINE KINASE)
NM_002938*	RNF4	RING FINGER PROTEIN 4
NM_002944*	ROS1	V-ROS UR2 SARCOMA VIRUS ONCOGENE HOMOLOG
		1 (AVIAN)
NM_002946*	RPA2	REPLICATION PROTEIN A2, 32 KDA
NM_005053*	RAD23A	RAD23 HOMOLOG A (S. CEREVISIAE)
NM_005228*	EGFR	EPIDERMAL GROWTH FACTOR RECEPTOR
		(ERYTHROBLASTIC LEUKEMIA VIRAL (V-ERB-B)
		ONCOGENE HOMOLOG, AVIAN)
NM_012478*	WBP2	WW DOMAIN BINDING PROTEIN 2
NM_017949*	CUEDC1	CUE DOMAIN CONTAINING 1
NM_020182*	TMEPAI	TRANSMEMBRANE, PROSTATE ANDROGEN
		INDUCED RNA
NM_020630*	RET	RET PROTO-ONCOGENE (MULTIPLE ENDOCRINE
		NEOPLASIA AND MEDULLARY THYROID
		CARCINOMA 1, HIRSCHSPRUNG DISEASE)
NM_030636*	EEPD1	KIAA1706 PROTEIN
NM_130465*	TSPAN17	TETRASPANIN 17
NM_152267*	RNF185	RING FINGER PROTEIN 185
NM_153229*	TMEM92	TRANSMEMBRANE PROTEIN 92
NM_153345*	TMEM139	HYPOTHETICAL PROTEIN FLJ90586
NM_194271*	RNF34	RING FINGER PROTEIN 34

Sumo2/3

NM_014805	EPM2AIP1	EPM2A (LAFORIN) INTERACTING PROTEIN 1
NM_177974	CASC4	CANCER SUSCEPTIBILITY CANDIDATE 4
BC017789	CHORDC1	CYSTEINE AND HISTIDINE-RICH DOMAIN (CHORD)-
		CONTAINING 1
NM_018393	TCP11L1	T-COMPLEX 11 (MOUSE) LIKE 1
NM_017588	WDR5	WD REPEAT DOMAIN 5
BC056402	LOC144097	HYPOTHETICAL PROTEIN BC007540
NM_003697	OR5F1	OLFACTORY RECEPTOR, FAMILY 5, SUBFAMILY F,
		MEMBER 1
NM_014868	RNF10	RING FINGER PROTEIN 10
NM_016269	LEF1	LYMPHOID ENHANCER-BINDING FACTOR 1
BC014475	BIRC7	LIVIN INHIBITOR-OF-APOTOSIS
BC009207	HIC2	HYPERMETHYLATED IN CANCER 2
NM_031845	MAP2	MICROTUBULE-ASSOCIATED PROTEIN 2
BC020523	INTS7	CHROMOSOME 1 OPEN READING FRAME 73
NM_018679	TCP11	T-COMPLEX 11 (MOUSE)
NM_019087	ARL15	ADP-RIBOSYLATION FACTOR-LIKE 15
BC043247	TLE3	TRANSDUCIN-LIKE ENHANCER OF SPLIT 3 (E(SP1)
		HOMOLOG, DROSOPHILA)
BC002677	AHDC1	AT HOOK, DNA BINDING MOTIF, CONTAINING 1
NM_003403	YY1	YY1 TRANSCRIPTION FACTOR
BC039583	MGEA5	MENINGIOMA EXPRESSED ANTIGEN 5
		(HYALURONIDASE)
NM_015148	PASK	PAS DOMAIN CONTAINING SERINE/THREONINE
		KINASE
BC010125	C3ORF37	CHROMOSOME 3 OPEN READING FRAME 37
NM_001786	CDC2	CELL DIVISION CYCLE 2, G1 TO S AND G2 TO M
BC005008	CEACAM6	CARCINOEMBRYONIC ANTIGEN-RELATED CELL
		ADHESION MOLECULE 6 (NON-SPECIFIC CROSS
		REACTING ANTIGEN)
NM_144706	C2ORF15	CHROMOSOME 2 OPEN READING FRAME 15
NM_007277	EXOC3	EXOCYST COMPLEX COMPONENT 3
NM_002648	PIM1	PIM-1 ONCOGENE
NM_002019	FLT1	FMS-RELATED TYROSINE KINASE 1 (VASCULAR
		ENDOTHELIAL GROWTH FACTOR/VASCULAR
		PERMEABILITY FACTOR RECEPTOR)
NM_152619	DCLK2	DOUBLECORTIN AND CAM KINASE-LIKE 2
BC022253	SLC6A15	SOLUTE CARRIER FAMILY 6, MEMBER 15
NM_017949	CUEDC1	CUE DOMAIN CONTAINING 1
NM_006002	UCHL3	UBIQUITIN CARBOXYL-TERMINAL ESTERASE L3
		(UBIQUITIN THIOLESTERASE)
NM_001278	CHUK	CONSERVED HELIX-LOOP-HELIX UBIQUITOUS
		KINASE
NM_001219	CALU	CALUMENIN
BC050645	BYSL	BYSTIN-LIKE
BC040272	IL16	INTERLEUKIN 16 (LYMPHOCYTE
		CHEMOATTRACTANT FACTOR)
BC023152	GYG2	GLYCOGENIN 2
NM_002011	FGFR4	FIBROBLAST GROWTH FACTOR RECEPTOR 4
BC024725	ANKRD50	ANKYRIN REPEAT DOMAIN 50
NM_138353	LOC90379	HYPOTHETICAL PROTEIN BC002926
BC061697	C3ORF62	CHROMOSOME 3 OPEN READING FRAME 62
NM_015417	SPEF1	CHROMOSOME 20 OPEN READING FRAME 28
NM_181707	C17ORF64	CHROMOSOME 17 OPEN READING FRAME 64
NM_199334	THRA	THYROID HORMONE RECEPTOR, ALPHA
		(ERYTHROBLASTIC LEUKEMIA VIRAL (V-ERB-A)
		ONCOGENE HOMOLOG, AVIAN)
BC060760	GIMAP6	IMMUNE ASSOCIATED NUCLEOTIDE 2
NM_002738	PRKCB1	PROTEIN KINASE C, BETA 1
BC000247	THAP4	THAP DOMAIN CONTAINING 4
BC013567	USP48	HYPOTHETICAL PROTEIN FLJ11328
NM_198498	C11ORF53	CHROMOSOME 11 OPEN READING FRAME 53
BC012289	KIAA0515	KIAA0515 PROTEIN
BC004219	AGPAT3	1-ACYLGLYCEROL-3-PHOSPHATE O-
		ACYLTRANSFERASE 3
NM_130766	SKIP	SKELETAL MUSCLE AND KIDNEY ENRICHED
		INOSITOL PHOSPHATASE
NM_001328	CTBP1	C-TERMINAL BINDING PROTEIN 1
BC058861	SULT1C4	SULFOTRANSFERASE FAMILY, CYTOSOLIC, 1C,
		MEMBER 2
BC046117	DNALI1	DYNEIN, AXONEMAL, LIGHT INTERMEDIATE
		POLYPEPTIDE 1
NM_032017	STK40	SERINE/THREONINE KINASE 40
NM_173822	FAM126B	HYPOTHETICAL PROTEIN MGC39518
BC032120	C20ORF11	CHROMOSOME 20 OPEN READING FRAME 11
NM_001556	IKBKB	INHIBITOR OF KAPPA LIGHT POLYPEPTIDE GENE
		ENHANCER IN B-CELLS, KINASE BETA
NM_032014	MRPS24	MITOCHONDRIAL RIBOSOMAL PROTEIN S24
NM_145796	POGZ	POGO TRANSPOSABLE ELEMENT WITH ZNF
		DOMAIN
NM_001042599	ERBB4
NM_017629	EIF2C4	ARGONAUTE 4
NM_032846	RAB2B	RAB2B, MEMBER RAS ONCOGENE FAMILY
BC011234	SMNDC1	SURVIVAL MOTOR NEURON DOMAIN CONTAINING 1
NM_017583	TRIM44	TRIPARTITE MOTIF-CONTAINING 44
NM_005639	SYT1	SYNAPTOTAGMIN I
NM_016954	TBX22	T-BOX 22
NM_002796	PSMB4	PROTEASOME (PROSOME, MACROPAIN) SUBUNIT,
		BETA TYPE, 4
NM_000666	ACY1	AMINOACYLASE 1
NM_032326	TMEM175	HYPOTHETICAL PROTEIN MGC4618
NM_001197	BIK	BCL2-INTERACTING KILLER (APOPTOSIS-
		INDUCING)
NM_170672	RASGRP3	RAS GUANYL RELEASING PROTEIN 3 (CALCIUM
		AND DAG-REGULATED)
BC017357	ZNF765	HYPOTHETICAL PROTEIN BC001610
BC020233	IGLC2	IMMUNOGLOBULIN LAMBDA CONSTANT 1 (MCG
		MARKER)
BC059374	STK31	SERINE/THREONINE KINASE 31
NM_014248	RBX1	RING-BOX 1
NM_005158	ABL2	V-ABL ABELSON MURINE LEUKEMIA VIRAL
		ONCOGENE HOMOLOG 2 (ARG, ABELSON-RELATED
		GENE)
NM_018668	VPS33B	VACUOLAR PROTEIN SORTING 33B (YEAST)
BC063451	TCP10L2	T-COMPLEX 10 (MOUSE)
NM_002623	PFDN4	PREFOLDIN SUBUNIT 4
BC016652	BMX	BMX NON-RECEPTOR TYROSINE KINASE
NM_153486	LDHD	LACTATE DEHYDROGENASE D
NM_033307	CASP4	CASPASE 4, APOPTOSIS-RELATED CYSTEINE
		PEPTIDASE
NM_004113	FGF12	FIBROBLAST GROWTH FACTOR 12
NM_005148	UNC119	UNC-119 HOMOLOG (C. ELEGANS)
NM_004838	HOMER3	HOMER HOMOLOG 3 (DROSOPHILA)
NM_016355	DDX47	DEAD (ASP-GLU-ALA-ASP) BOX POLYPEPTIDE 47
NM_014548	TMOD2	TROPOMODULIN 2 (NEURONAL)
BC016964	MRGPRF	MAS-RELATED GPR, MEMBER F
BC029220	SOX5	SRY (SEX DETERMINING REGION Y)-BOX 5
BC030711	C2ORF13	CHROMOSOME 2 OPEN READING FRAME 13
NM_001571	IRF3	INTERFERON REGULATORY FACTOR 3
BC031830	KLHL32	KIAA1900
NM_153498	CAMK1D	CALCIUM/CALMODULIN-DEPENDENT PROTEIN
		KINASE ID
NM_144602	C16ORF78	HYPOTHETICAL PROTEIN MGC32905
NM_012325	MAPRE1	MICROTUBULE-ASSOCIATED PROTEIN, RP/EB
		FAMILY, MEMBER 1
BC057840	PSMB5	PROTEASOME (PROSOME, MACROPAIN) SUBUNIT,
		BETA TYPE, 5
NM_079422	MYL1	MYOSIN, LIGHT POLYPEPTIDE 1, ALKALI;
		SKELETAL, FAST
BC029267	MUC20	MUCIN 20
NM_020830	WDFY1	WD REPEAT AND FYVE DOMAIN CONTAINING 1
NM_033003	GTF2I
BC009571	STRA13	STIMULATED BY RETINOIC ACID 13 HOMOLOG
		(MOUSE)
NM_005030	PLK1	POLO-LIKE KINASE 1 (DROSOPHILA)
NM_022754	SFXN1	LIKELY ORTHOLOG OF MOUSE SIDEROFLEXIN 1
BC012997	SULF1	SULFATASE 1
NM_001221	CAMK2D	CALCIUM/CALMODULIN-DEPENDENT PROTEIN
		KINASE (CAM KINASE) II DELTA
BC031691	SLAIN2	KIAA1458 PROTEIN
NM_014840	NUAK1	NUAK FAMILY, SNF1-LIKE KINASE, 1
BC001772	QARS	GLUTAMINYL-TRNA SYNTHETASE
NM_032693	ARD1B
BC025314	IGHG1	IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 1
		(G1M MARKER)
BC033491	ADAD2	TESTIS NUCLEAR RNA-BINDING PROTEIN-LIKE
BC009650	PDS5A	SCC-112 PROTEIN
NM_018326	GIMAP4	GTPASE, IMAP FAMILY MEMBER 4
NM_005239	ETS2	V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENE
		HOMOLOG 2 (AVIAN)
NM_006257	PRKCQ	PROTEIN KINASE C, THETA
NM_152667	NANP	N-ACETYLNEURAMINIC ACID PHOSPHATASE
BC001728*	TFPT	TCF3 (E2A) FUSION PARTNER (IN CHILDHOOD
		LEUKEMIA)
BC001772*	QARS	GLUTAMINYL-TRNA SYNTHETASE
BC007048*	ZMYM5	ZINC FINGER, MYM-TYPE 5
BC010125*	C3ORF37	CHROMOSOME 3 OPEN READING FRAME 37
BC017314*	ETS1	V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENE
		HOMOLOG 1 (AVIAN)
BC020985*	COASY	COENZYME A SYNTHASE
BC036572*	ZCCHC12	ZINC FINGER, CCHC DOMAIN CONTAINING 12
BC040949*	MEF2D	MADS BOX TRANSCRIPTION ENHANCER FACTOR 2,
		POLYPEPTIDE D (MYOCYTE ENHANCER FACTOR
		2D)
BC056402*	LOC144097	HYPOTHETICAL PROTEIN BC007540
BC056415*	RPAP3	HYPOTHETICAL PROTEIN FLJ21908
NM_001014796*	DDR2	DISCOIDIN DOMAIN RECEPTOR FAMILY, MEMBER 2
NM_001039468*	MARK2	MAP/MICROTUBULE AFFINITY-REGULATING
		KINASE 2
NM_001786*	CDC2	CELL DIVISION CYCLE 2, G1 TO S AND G2 TO M
NM_001910*	CTSE	CATHEPSIN E
NM_002378*	MATK	MEGAKARYOCYTE-ASSOCIATED TYROSINE
		KINASE
NM_002497*	NEK2	NIMA (NEVER IN MITOSIS GENE A)-RELATED
		KINASE 2
NM_002938*	RNF4	RING FINGER PROTEIN 4
NM_003141*	TRIM21	TRIPARTITE MOTIF-CONTAINING 21
NM_006257*	PRKCQ	PROTEIN KINASE C, THETA
NM_006259*	PRKG2	PROTEIN KINASE, CGMP-DEPENDENT, TYPE II
NM_006937*	SUMO2	SMT3 SUPPRESSOR OF MIF TWO 3 HOMOLOG 2
		(YEAST)
NM_015981*	CAMK2A	CALCIUM/CALMODULIN-DEPENDENT PROTEIN
		KINASE (CAM KINASE) II ALPHA
NM_016058*	TPRKB	TP53RK BINDING PROTEIN
NM_017838*	NOLA2	NUCLEOLAR PROTEIN FAMILY A, MEMBER 2
		(H/ACA SMALL NUCLEOLAR RNPS)
NM_021709*	SIVA1	CD27-BINDING (SIVA) PROTEIN
NM_032752*	ZNF496	ZINC FINGER PROTEIN 496
NM_130807*	MOBKL2A	MOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE
		2A (YEAST)
NM_145173*	DIRAS1	DIRAS FAMILY, GTP-BINDING RAS-LIKE 1
NM_175907*	ZADH2	HYPOTHETICAL PROTEIN BC010734
NM_033003*	NA	NA

Nedd8

BC000178	KCMF1	POTASSIUM CHANNEL MODULATORY FACTOR 1
BC000395	LETMD1	LETM1 DOMAIN CONTAINING 1
BC001852	THG1L	INTERPHASE CYCTOPLASMIC FOCI PROTEIN 45
BC002526	HSPA4	HEAT SHOCK 70 KDA PROTEIN 4
BC007312	KIRREL2	KIN OF IRRE LIKE 2 (DROSOPHILA)
BC009074	C8ORF70	CHROMOSOME 8 OPEN READING FRAME 70
BC009485	C4ORF16	CHROMOSOME 4 OPEN READING FRAME 16
BC012945	C19ORF57	HYPOTHETICAL PROTEIN MGC11271
BC018953	SMARCD2	SWI/SNF RELATED, MATRIX ASSOCIATED, ACTIN
		DEPENDENT REGULATOR OF CHROMATIN,
		SUBFAMILY D, MEMBER 2
BC020658	TMEM40	TRANSMEMBRANE PROTEIN 40
BC038504	SNF1LK	SNF1-LIKE KINASE
BC050696	C12ORF48	CHROMOSOME 12 OPEN READING FRAME 48
BC051849	RPAIN	RPA INTERACTING PROTEIN
BC062736	CTD-2090I13.4	BASIC TRANSCRIPTION FACTOR 3, PSEUDOGENE 9
NM_004235	KLF4	KRUPPEL-LIKE FACTOR 4 (GUT)
NM_004391	CYP8B1	CYTOCHROME P450, FAMILY 8, SUBFAMILY B,
		POLYPEPTIDE 1
NM_005206	CRK	V-CRK SARCOMA VIRUS CT10 ONCOGENE
		HOMOLOG (AVIAN)
NM_005651	TDO2	TRYPTOPHAN 2,3-DIOXYGENASE
NM_006251	PRKAA1	PROTEIN KINASE, AMP-ACTIVATED, ALPHA 1
		CATALYTIC SUBUNIT
NM_012328	DNAJB9	DNAJ (H5P40) HOMOLOG, SUBFAMILY B, MEMBER 9
NM_013442	STOML2	STOMATIN (EPB72)-LIKE 2
NM_014878	KIAA0020	KIAA0020
NM_018014	BCL11A	B-CELL CLL/LYMPHOMA 11A (ZINC FINGER
		PROTEIN)
NM_019895	CLNS1A	CHLORIDE CHANNEL, NUCLEOTIDE-SENSITIVE, 1A
NM_021803	IL21	INTERLEUKIN 21
NM_152443	RDH12	RETINOL DEHYDROGENASE 12 (ALL-TRANS AND 9-
		CIS)
BC051366	NA	NA
BC005008*	CEACAM6	CARCINOEMBRYONIC ANTIGEN-RELATED CELL
		ADHESION MOLECULE 6 (NON-SPECIFIC CROSS
		REACTING ANTIGEN)
BC006323*	ABCB7	ATP-BINDING CASSETTE, SUB-FAMILY B
		(MDR/TAP), MEMBER 7
BC011707*	NRBF2	NUCLEAR RECEPTOR BINDING FACTOR 2
BC012109*	HOMER2	HOMER HOMOLOG 2 (DROSOPHILA)
BC020985*	COASY	COENZYME A SYNTHASE
BC021906*	FMNL1	FORMIN-LIKE 1
BC053895*	IRS1	INSULIN RECEPTOR SUBSTRATE 1
BC056669*	DCUN1D2	DCN1, DEFECTIVE IN CULLIN NEDDYLATION 1,
		DOMAIN CONTAINING 2 (S. CEREVISIAE)
BC058924*	UBE2M	UBIQUITIN-CONJUGATING ENZYME E2M (UBC12
		HOMOLOG, YEAST)
NM_001004105*	GRK6	G PROTEIN-COUPLED RECEPTOR KINASE 6
NM_001039468*	MARK2	MAP/MICROTUBULE AFFINITY-REGULATING
		KINASE 2
NM_001798*	CDK2	CYCLIN-DEPENDENT KINASE 2
NM_001895*	CSNK2A1	CASEIN KINASE 2, ALPHA 1 POLYPEPTIDE
NM_003141*	TRIM21	TRIPARTITE MOTIF-CONTAINING 21
NM_003668*	MAPKAPK5	MITOGEN-ACTIVATED PROTEIN KINASE-
		ACTIVATED PROTEIN KINASE 5
NM_005019*	PDE1A	PHOSPHODIESTERASE 1A, CALMODULIN-
		DEPENDENT
NM_005038*	PPID	PEPTIDYLPROLYL ISOMERASE D (CYCLOPHILIN D)
NM_006156*	NEDD8	NEURAL PRECURSOR CELL EXPRESSED,
		DEVELOPMENTALLY DOWN-REGULATED 8
NM_012247*	SEPHS1	SELENOPHOSPHATE SYNTHETASE 1
NM_012325*	MAPRE1	MICROTUBULE-ASSOCIATED PROTEIN, RP/EB
		FAMILY, MEMBER 1
NM_015417*	SPEF1	CHROMOSOME 20 OPEN READING FRAME 28
NM_016058*	TPRKB	TP53RK BINDING PROTEIN
NM_018014*	BCL11A	B-CELL CLL/LYMPHOMA 11A (ZINC FINGER
		PROTEIN)
NM_022754*	SFXN1	LIKELY ORTHOLOG OF MOUSE SIDEROFLEXIN 1
NM_030662*	MAP2K2	MITOGEN-ACTIVATED PROTEIN KINASE KINASE 2
NM_032141*	CCDC55	COILED-COIL DOMAIN CONTAINING 55
NM_130439*	MXI1	MAX INTERACTOR 1
NM_138559*	BCL11A	B-CELL CLL/LYMPHOMA 11A (ZINC FINGER
		PROTEIN)
NM_175907*	ZADH2	HYPOTHETICAL PROTEIN BC010734
NM_212535*	PRKCB1	PROTEIN KINASE C, BETA 1

FAT10

NM_005737	ARL4C	ADP-RIBOSYLATION FACTOR-LIKE 4C
BC013648	EFHD2	EF-HAND DOMAIN FAMILY, MEMBER D2
BC031247	CCDC67	COILED-COIL DOMAIN CONTAINING 67
NM_015621	CCDC69	COILED-COIL DOMAIN CONTAINING 69
NM_024099	C11ORF48	CHROMOSOME 11 OPEN READING FRAME 48
NM_016951	CKLF	CHEMOKINE-LIKE FACTOR
BC008919	TBC1D9B	KIAA0676 PROTEIN
NM_032855	HSH2D	HEMATOPOIETIC SH2 DOMAIN CONTAINING
NM_152788	ANKS1B	ANKYRIN REPEAT AND STERILE ALPHA MOTIF
		DOMAIN CONTAINING 1B
NM_001277	CHKA	CHOLINE KINASE ALPHA
NM_152434	CWF19L2	CWF19-LIKE 2, CELL CYCLE CONTROL (S. POMBE)
NM_004811	LPXN	LEUPAXIN
NM_182739	NDUFB6	NADH DEHYDROGENASE (UBIQUINONE) 1 BETA
		SUBCOMPLEX, 6, 17 KDA
BC053602	C15ORF38	HYPOTHETICAL PROTEIN FLJ35955
NM_018976	SLC38A2	SOLUTE CARRIER FAMILY 38, MEMBER 2
BC004967	UBAC1	UBIQUITIN ASSOCIATED DOMAIN CONTAINING 1
BC010360	LMBRD1	LMBR1 DOMAIN CONTAINING 1
BC016381	NA	HYPOTHETICAL PROTEIN
BC017101	POMZP3	POM (POM121 HOMOLOG, RAT) AND ZP3 FUSION
BC026175	ATF2	ACTIVATING TRANSCRIPTION FACTOR 2
BC062359	C8ORF47	CHROMOSOME 8 OPEN READING FRAME 47
NM_000301	PLG	PLASMINOGEN
NM_002815	PSMD11	PROTEASOME (PROSOME, MACROPAIN) 26S
		SUBUNIT, NON-ATPASE, 11
NM_002854	PVALB	PARVALBUMIN
NM_012198	GCA	GRANCALCIN, EF-HAND CALCIUM BINDING
		PROTEIN
NM_017727	FLJ20254	HYPOTHETICAL PROTEIN FLJ20254
NM_021925	C2ORF43	HYPOTHETICAL PROTEIN FLJ21820
NM_138785	C6ORF72	CHROMOSOME 6 OPEN READING FRAME 72
NM_144686	TMC4	TRANSMEMBRANE CHANNEL-LIKE 4
NM_012416	RANBP6	RAN BINDING PROTEIN 6
NM_006899	IDH3B	ISOCITRATE DEHYDROGENASE 3 (NAD+) BETA
BC001726	NOL11	NUCLEOLAR PROTEIN 11
BC015219	RBCK1	CHROMOSOME 20 OPEN READING FRAME 18
BC034801	ZDHHC19	ZINC FINGER, DHHC-TYPE CONTAINING 19
BC022244	PYCR1	PYRROLINE-5-CARBOXYLATE REDUCTASE 1
NM_006399	BATF	BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR,
		ATF-LIKE
BC014949	DHX58	LIKELY ORTHOLOG OF MOUSE D11LGP2
NM_014182	ORMDL2	ORM1-LIKE 2 (S. CEREVISIAE)
NM_024114	TRIM48	TRIPARTITE MOTIF-CONTAINING 48
NM_006607	PTTG2	PITUITARY TUMOR-TRANSFORMING 2
NM_004357	CD151	CD151 ANTIGEN (RAPH BLOOD GROUP)
NM_005513	GTF2E1	GENERAL TRANSCRIPTION FACTOR IIE,
		POLYPEPTIDE 1, ALPHA 56 KDA
NM_016231	NLK	NEMO-LIKE KINASE
NM_054033	FKBP1B	FK506 BINDING PROTEIN 1B, 12.6 KDA
NM_152646		hypothetical protein MGC23270
NM_173518	C8ORF45	CHROMOSOME 8 OPEN READING FRAME 45
NM_177951	PPM1A	PROTEIN PHOSPHATASE 1A (FORMERLY 2C),
		MAGNESIUM-DEPENDENT, ALPHA ISOFORM
NM_020990	CKMT1B	CREATINE KINASE, MITOCHONDRIAL 1B
NM_001258	CDK3	CYCLIN-DEPENDENT KINASE 3
NM_138565	CTTN	CORTACTIN
NM_018189	DPPA4	DEVELOPMENTAL PLURIPOTENCY ASSOCIATED 4
NM_001330	CTF1	CARDIOTROPHIN 1
BC029541	LETM2	LEUCINE ZIPPER-EF-HAND CONTAINING
		TRANSMEMBRANE PROTEIN 2
NM_144594	GTSF1	FAMILY WITH SEQUENCE SIMILARITY 112,
		MEMBER B
NM_173192	KCNIP2	KV CHANNEL INTERACTING PROTEIN 2
BC034468	FLJ11171	HYPOTHETICAL PROTEIN FLJ11171
NM_033306	CASP4	CASPASE 4, APOPTOSIS-RELATED CYSTEINE
		PEPTIDASE
BC041132	KIFC3	KINESIN FAMILY MEMBER C3
BC011461	MITF	MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION
		FACTOR
BC046214	MPHOSPH8	M-PHASE PHOSPHOPROTEIN, MPP8
BC057774	RG9MTD3	RNA (GUANINE-9-) METHYLTRANSFERASE DOMAIN
		CONTAINING 3
NM_016606	REEP2	RECEPTOR ACCESSORY PROTEIN 2
NM_145265	CCDC127	SIMILAR TO RIKEN CDNA 0610011N22
BC015056	ACAD10	ACYL-COENZYME A DEHYDROGENASE FAMILY,
		MEMBER 10
BC007224	GALNT10	UDP-N-ACETYL-ALPHA-D-
		GALACTOSAMINE:POLYPEPTIDE N-
		ACETYLGALACTOSAMINYLTRANSFERASE 10
		(GALNAC-T10)
BC009289	ACSBG1	ACYL-COA SYNTHETASE BUBBLEGUM FAMILY
		MEMBER 1
BC011786	NA	CHROMOSOME 11 OPEN READING FRAME 43
NM_000559	HBG2	HEMOGLOBIN, GAMMA A
NM_024680	E2F8	E2F TRANSCRIPTION FACTOR 8
BC000557	PEMT	PHOSPHATIDYLETHANOLAMINE N-
		METHYLTRANSFERASE
BC005974	VAMP4	VESICLE-ASSOCIATED MEMBRANE PROTEIN 4
BC009771	BCCIP	CDK INHIBITOR P21 BINDING PROTEIN
BC053508	ARL6IP2	ADP-RIBOSYLATION FACTOR-LIKE 6 INTERACTING
		PROTEIN 2
NM_001307	CLDN7	CLAUDIN 7
NM_002688	12:00 AM	SEPTIN 5
NM_004123	GIP	GASTRIC INHIBITORY POLYPEPTIDE
NM_004545	NDUFB1	NADH DEHYDROGENASE (UBIQUINONE) 1 BETA
		SUBCOMPLEX, 1, 7 KDA
NM_004712	HGS	HEPATOCYTE GROWTH FACTOR-REGULATED
		TYROSINE KINASE SUBSTRATE
NM_005621	S100A12	S100 CALCIUM BINDING PROTEIN A12
		(CALGRANULIN C)
NM_016388	TRAT1	T CELL RECEPTOR ASSOCIATED TRANSMEMBRANE
		ADAPTOR 1
NM_138998	DDX39	DEAD (ASP-GLU-ALA-ASP) BOX POLYPEPTIDE 39
NM_144673	CMTM2	CKLF-LIKE MARVEL TRANSMEMBRANE DOMAIN
		CONTAINING 2
NM_182597	C7ORF53	HYPOTHETICAL PROTEIN FLJ39575
BC035601	WWC3	KIAA1280 PROTEIN
BC036365	C10ORF81	HYPOTHETICAL PROTEIN LOC338564
NM_002103	GYS1	GLYCOGEN SYNTHASE 1 (MUSCLE)
NM_145252	LOC124220	SIMILAR TO COMMON SALIVARY PROTEIN 1
NM_139280	ORMDL3	HYPOTHETICAL PROTEIN LOC51242
NM_022372	GBL	G PROTEIN BETA SUBUNIT-LIKE
BC052805	EPB49	ERYTHROCYTE MEMBRANE PROTEIN BAND 4.9
		(DEMATIN)
NM_014551	NCAPH2	KLEISIN BETA
NM_017848	FAM120C	CHROMOSOME X OPEN READING FRAME 17
BC008141	UCHL5IP	THREE PRIME REPAIR EXONUCLEASE 2
NM_005832	KCNMB2	POTASSIUM LARGE CONDUCTANCE CALCIUM-
		ACTIVATED CHANNEL, SUBFAMILY M, BETA
		MEMBER 2
NM_173517	VKORC1L1	VITAMIN K EPOXIDE REDUCTASE COMPLEX,
		SUBUNIT 1-LIKE 1
NM_173473	C10ORF104	CHROMOSOME 10 OPEN READING FRAME 104
NM_030650	KIAA1715	KIAA1715
NM_014570	ARFGAP3	ADP-RIBOSYLATION FACTOR GTPASE ACTIVATING
		PROTEIN 3
NM_021159	RAP1GDS1	RAP1, GTP-GDP DISSOCIATION STIMULATOR 1
BC017066	PRRC1	HYPOTHETICAL PROTEIN MGC12103
NM_014805	EPM2AIP1	EPM2A (LAFORIN) INTERACTING PROTEIN 1
BC033734	C17ORF66	CHROMOSOME 17 OPEN READING FRAME 66
NM_021644	HNRPH3	HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN
		H3 (2H9)
BC021987	NMI	N-MYC (AND STAT) INTERACTOR
NM_002489	NDUFA4	NADH DEHYDROGENASE (UBIQUINONE) 1 ALPHA
		SUBCOMPLEX, 4, 9 KDA
NM_033542	DBNDD2	CHROMOSOME 20 OPEN READING FRAME 35
BC015754	CADPS	CA2+-DEPENDENT SECRETION ACTIVATOR
NM_032357	CCDC115	HYPOTHETICAL PROTEIN MGC12981
XM_291436
BC012266	ATG12	ATG12 AUTOPHAGY RELATED 12 HOMOLOG (S. CEREVISIAE)
BC012377	EGFL7	EGF-LIKE-DOMAIN, MULTIPLE 7
BC017943	PPP1R1C	PROTEIN PHOSPHATASE 1, REGULATORY
		(INHIBITOR) SUBUNIT 1C
BC058031	HP	HAPTOGLOBIN
BC060828	ARID3A	AT RICH INTERACTIVE DOMAIN 3A (BRIGHT-LIKE)
NM_144586	LYPD1	LY6/PLAUR DOMAIN CONTAINING 1
BC009106	SEC16B	LEUCINE ZIPPER TRANSCRIPTION REGULATOR 2
NM_018990	CXORF9	CHROMOSOME X OPEN READING FRAME 9
NM_004935	CDK5	CYCLIN-DEPENDENT KINASE 5
BC014484	TOR1A	TORSIN FAMILY 1, MEMBER A (TORSIN A)
BC063111	GGT6	GAMMA-GLUTAMYLTRANSFERASE 6 HOMOLOG
		(RAT)
NM_023937	MRPL34	MITOCHONDRIAL RIBOSOMAL PROTEIN L34
NM_030810	TXNDC5	THIOREDOXIN DOMAIN CONTAINING 5
NM_138463	TLCD1	TLC DOMAIN CONTAINING 1
BC007919	STARD10	START DOMAIN CONTAINING 10
BC016703	ACSM5	HYPOTHETICAL PROTEIN FLJ20581
NM_001004354	NRARP	SIMILAR TO ANKYRIN-REPEAT PROTEIN NRARP
NM_002436	MPP1	MEMBRANE PROTEIN, PALMITOYLATED 1, 55 KDA
NM_004013	DMD	DYSTROPHIN (MUSCULAR DYSTROPHY, DUCHENNE
		AND BECKER TYPES)
NM_018335	C14ORF131	CHROMOSOME 14 OPEN READING FRAME 131
NM_138385	TMEM129	TRANSMEMBRANE PROTEIN 129
NM_001823	CKB	CREATINE KINASE, BRAIN
NM_004440	EPHA7	EPH RECEPTOR A7
NM_006779	CDC42EP2	CDC42 EFFECTOR PROTEIN (RHO GTPASE BINDING) 2
NM_007162	TFEB	TRANSCRIPTION FACTOR EB
NM_014248	RBX1	RING-BOX 1
NM_016267	VGLL1	VESTIGIAL LIKE 1 (DROSOPHILA)
NM_181656	C17ORF58	CHROMOSOME 17 OPEN READING FRAME 58
NM_138482		hypothetical protein BC009264
BC026345	KIAA1189	KIAA1189
NM_032315	SLC25A33	PNC1 PROTEIN
NM_002944	ROS1	V-ROS UR2 SARCOMA VIRUS ONCOGENE HOMOLOG
		1 (AVIAN)
BC017048	GJB2	GAP JUNCTION PROTEIN, BETA 2, 26 KDA
		(CONNEXIN 26)
BC039814	ZRANB2	ZINC FINGER PROTEIN 265
NM_001044	SLC6A3	SOLUTE CARRIER FAMILY 6 (NEUROTRANSMITTER
		TRANSPORTER, DOPAMINE), MEMBER 3
NM_138470		hypothetical protein BC008131
NM_005084	PLA2G7	PHOSPHOLIPASE A2, GROUP VII (PLATELET-
		ACTIVATING FACTOR ACETYLHYDROLASE,
		PLASMA)
BC012499	SIRT1	SIRTUIN (SILENT MATING TYPE INFORMATION
		REGULATION 2 HOMOLOG) 1 (S. CEREVISIAE)
BC045532	LSM8	LSM8 HOMOLOG, U6 SMALL NUCLEAR RNA
		ASSOCIATED (S. CEREVISIAE)
NM_003295	TPT1	TUMOR PROTEIN, TRANSLATIONALLY-
		CONTROLLED 1
NM_006912	RIT1	RAS-LIKE WITHOUT CAAX 1
NM_014184	CNIH4	CORNICHON HOMOLOG 4 (DROSOPHILA)
BC003065	CDK2	CYCLIN-DEPENDENT KINASE 2
BC009793	ERCC8	EXCISION REPAIR CROSS-COMPLEMENTING
		RODENT REPAIR DEFICIENCY, COMPLEMENTATION
		GROUP 8
NM_005114	HS3ST1	HEPARAN SULFATE (GLUCOSAMINE) 3-O-
		SULFOTRANSFERASE 1
NM_018129	PNPO	PYRIDOXINE 5′-PHOSPHATE OXIDASE
NM_152285	ARRDC1	ARRESTIN DOMAIN CONTAINING 1
BC009710	GOSR2	GOLGI SNAP RECEPTOR COMPLEX MEMBER 2
NM_015966	ERGIC3	ERGIC AND GOLGI 3
NM_020370	GPR84	G PROTEIN-COUPLED RECEPTOR 84
NM_130398	EXO1	EXONUCLEASE 1
NM_145865	ANKS4B	ANKYRIN REPEAT AND STERILE ALPHA MOTIF
		DOMAIN CONTAINING 4B
BC001234	LOH11CR2A	LOSS OF HETEROZYGOSITY, 11, CHROMOSOMAL
		REGION 2, GENE A
BC062625	SLC39A4	SOLUTE CARRIER FAMILY 39 (ZINC TRANSPORTER),
		MEMBER 4
BC001889	NAPG	N-ETHYLMALEIMIDE-SENSITIVE FACTOR
		ATTACHMENT PROTEIN, GAMMA
BC013768	PCCB	PROPIONYL COENZYME A CARBOXYLASE, BETA
		POLYPEPTIDE
BC020651	MRPL35	MITOCHONDRIAL RIBOSOMAL PROTEIN L35
BC051291	RDH11	RETINOL DEHYDROGENASE 11 (ALL-TRANS AND 9-
		CIS)
BC069328	BMF	BCL2 MODIFYING FACTOR
NM_006426	DPYSL4	DIHYDROPYRIMIDINASE-LIKE 4
NM_178863	KCTD13	POTASSIUM CHANNEL TETRAMERISATION DOMAIN
		CONTAINING 13
BC004176	SSH3	SLINGSHOT HOMOLOG 3 (DROSOPHILA)
BC008790	GSTM3	GLUTATHIONE S-TRANSFERASE M3 (BRAIN)
BC010176	NY-SAR-48	SARCOMA ANTIGEN NY-SAR-48
BC020885	C12ORF65	HYPOTHETICAL PROTEIN FLJ38663
BC034554	SERPINA3	SERPIN PEPTIDASE INHIBITOR, CLADE A (ALPHA-1
		ANTIPROTEINASE, ANTITRYPSIN), MEMBER 3
NM_000394	CRYAA	CRYSTALLIN, ALPHA A
NM_078476	BTN2A1	BUTYROPHILIN, SUBFAMILY 2, MEMBER A1
BC015904	MRPL10	MITOCHONDRIAL RIBOSOMAL PROTEIN L10
BC019039	RGS3	REGULATOR OF G-PROTEIN SIGNALLING 3
BC067445	DAB1	DISABLED HOMOLOG 1 (DROSOPHILA)
NM_003221	TFAP2B	TRANSCRIPTION FACTOR AP-2 BETA (ACTIVATING
		ENHANCER BINDING PROTEIN 2 BETA)
NM_015959	TXNDC14	THIOREDOXIN DOMAIN CONTAINING 14
BC010033	QPRT	QUINOLINATE PHOSPHORIBOSYLTRANSFERASE
		(NICOTINATE-NUCLEOTIDE PYROPHOSPHORYLASE
		(CARBOXYLATING))
NM_152522	ARL6IP6	ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTING
		PROTEIN 6
BC019254	ENOX2	CYTOSOLIC OVARIAN CARCINOMA ANTIGEN 1
NM_012148	DUX3	DOUBLE HOMEOBOX, 3
NM_025004	CCDC15	COILED-COIL DOMAIN CONTAINING 15
BC017475	TTC15	TETRATRICOPEPTIDE REPEAT DOMAIN 15
NM_172211	CSF1	COLONY STIMULATING FACTOR 1 (MACROPHAGE)
BC007862	GPR108	G PROTEIN-COUPLED RECEPTOR 108
BC010850	HEATR2	HYPOTHETICAL PROTEIN FLJ20397
NM_016009	SH3GLB1	SH3-DOMAIN GRB2-LIKE ENDOPHILIN B1
NM_152328	ADSSL1	ADENYLOSUCCINATE SYNTHASE LIKE 1
BC020867	SLC6A13	SOLUTE CARRIER FAMILY 6 (NEUROTRANSMITTER
		TRANSPORTER, GABA), MEMBER 13
NM_178126	FAM134C	HYPOTHETICAL PROTEIN LOC162427
NM_007241	SNF8	SNF8, ESCRT-II COMPLEX SUBUNIT, HOMOLOG (S. CEREVISIAE)
NM_016440	VRK3	VACCINIA RELATED KINASE 3
BC035314	BXDC1	BRIX DOMAIN CONTAINING 1
NM_030881	DDX17	DEAD (ASP-GLU-ALA-ASP) BOX POLYPEPTIDE 17
NM_001033578	SGK3	SERUM/GLUCOCORTICOID REGULATED KINASE
		FAMILY, MEMBER 3
BC010155	FDX1L	SIMILAR TO RIKEN CDNA B230118G17 GENE
NM_018667	SMPD3	SPHINGOMYELIN PHOSPHODIESTERASE 3,
		NEUTRAL MEMBRANE (NEUTRAL
		SPHINGOMYELINASE II)
NM_017812	CHCHD3	COILED-COIL-HELIX-COILED-COIL-HELIX DOMAIN
		CONTAINING 3
NM_001613	ACTG2	ACTIN, ALPHA 2, SMOOTH MUSCLE, AORTA
BC031329	TMEM149	U2(RNU2) SMALL NUCLEAR RNA AUXILIARY
		FACTOR 1-LIKE 4
BC039256	PDS5B	ANDROGEN-INDUCED PROLIFERATION INHIBITOR
NM_017634	KCTD9	POTASSIUM CHANNEL TETRAMERISATION DOMAIN
		CONTAINING 9
NM_001017980	LOC203547	HYPOTHETICAL PROTEIN LOC203547
BC053320	CTBP1	C-TERMINAL BINDING PROTEIN 1
NM_152619	DCLK2	DOUBLECORTIN AND CAM KINASE-LIKE 2
BC033668	ARHGAP28	KIAA1314 PROTEIN
BC059396	FAM92A3	FAMILY WITH SEQUENCE SIMILARITY 92, MEMBER
		A3
NM_080660	ZC3HAV1L	SIMILAR TO RIKEN CDNA 1200014N16 GENE
BC003551	TGM2	TRANSGLUTAMINASE 2 (C POLYPEPTIDE, PROTEIN-
		GLUTAMINE-GAMMA-GLUTAMYLTRANSFERASE)
NM_172341	LIN37	PRESENILIN ENHANCER 2 HOMOLOG (C. ELEGANS)
NM_005158	ABL2	V-ABL ABELSON MURINE LEUKEMIA VIRAL
		ONCOGENE HOMOLOG 2 (ARG, ABELSON-RELATED
		GENE)
NM_005558	LAD1	LADININ 1
NM_000624	SERPINA5	SERPIN PEPTIDASE INHIBITOR, CLADE A (ALPHA-1
		ANTIPROTEINASE, ANTITRYPSIN), MEMBER 5
NM_173799	VSTM3	V-SET AND IMMUNOGLOBULIN DOMAIN
		CONTAINING 9
NM_003592	CUL1	CULLIN 1
BC017594	APIP	APAF1 INTERACTING PROTEIN
NM_032498	RHOXF2	PEPP SUBFAMILY GENE 2
BC008730	HK1	HEXOKINASE 1
BC016276	DLG7	DISCS, LARGE HOMOLOG 7 (DROSOPHILA)
BC033708	RALGPS1	RAL GEF WITH PH DOMAIN AND SH3 BINDING
		MOTIF 1
BC051000	TCL1B	T-CELL LEUKEMIA/LYMPHOMA 1B
BC066974	NA	HYPOTHETICAL PROTEIN
BC022983	LNX1	LIGAND OF NUMB-PROTEIN X 1
NM_003256	TIMP4	TIMP METALLOPEPTIDASE INHIBITOR 4
NM_003674	CDK10	CYCLIN-DEPENDENT KINASE (CDC2-LIKE) 10
BC004549	DUS3L	DIHYDROURIDINE SYNTHASE 3-LIKE (S. CEREVISIAE)
BC015596*	C21ORF51	CHROMOSOME 21 OPEN READING FRAME 51
BC018206*	FAM128B	HYPOTHETICAL PROTEIN FLJ14346
BC018722*	ASPSCR1	ALVEOLAR SOFT PART SARCOMA CHROMOSOME
		REGION, CANDIDATE 1
BC022357*	RPL17	RIBOSOMAL PROTEIN L17
BC023152*	GYG2	GLYCOGENIN 2
BC025700*	AFF4	AF4/FMR2 FAMILY, MEMBER 4
BC032825*	SH3GL2	SH3-DOMAIN GRB2-LIKE 2
BC038838*	PRR16	MESENCHYMAL STEM CELL PROTEIN DSC54
BC052805*	EPB49	ERYTHROCYTE MEMBRANE PROTEIN BAND 4.9
		(DEMATIN)
BC056415*	RPAP3	HYPOTHETICAL PROTEIN FLJ21908
BC065370*	C20ORF112	CHROMOSOME 20 OPEN READING FRAME 112
NM_001032296*	STK24	SERINE/THREONINE KINASE 24 (STE20 HOMOLOG,
		YEAST)
NM_002498*	NEK3	NIMA (NEVER IN MITOSIS GENE A)-RELATED
		KINASE 3
NM_002624*	PFDN5	PREFOLDIN SUBUNIT 5
NM_004329*	BMPR1A	BONE MORPHOGENETIC PROTEIN RECEPTOR, TYPE
		IA
NM_014245*	RNF7	RING FINGER PROTEIN 7
NM_014548*	TMOD2	TROPOMODULIN 2 (NEURONAL)
NM_015646*	RAP1B	RAP1B, MEMBER OF RAS ONCOGENE FAMILY
NM_017949*	CUEDC1	CUE DOMAIN CONTAINING 1
NM_018393*	TCP11L1	T-COMPLEX 11 (MOUSE) LIKE 1
NM_018679*	TCP11	T-COMPLEX 11 (MOUSE)
NM_024591*	CHMP6	CHROMATIN MODIFYING PROTEIN 6
NM_032368*	LZIC	LEUCINE ZIPPER AND CTNNBIP1 DOMAIN
		CONTAINING
NM_033118*	MYLK2	MYOSIN LIGHT CHAIN KINASE 2, SKELETAL
		MUSCLE
NM_130807*	MOBKL2A	MOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE
		2A (YEAST)
NM_145173*	DIRAS1	DIRAS FAMILY, GTP-BINDING RAS-LIKE 1
NM_152376*	UBXD3	UBX DOMAIN CONTAINING 3
NM_182493*	MLCK	MLCK PROTEIN
BC056907*	NA	NA

SUMO1

BC033766	NDUFV3	NADH DEHYDROGENASE (UBIQUINONE)
		FLAVOPROTEIN 3, 10 KDA
NM_001312	CRIP2	CYSTEINE-RICH PROTEIN 2
NM_004111	FEN1	FLAP STRUCTURE-SPECIFIC ENDONUCLEASE 1
NM_000805	GAST	GASTRIN
NM_030645	SH3BP5L	SH3-BINDING DOMAIN PROTEIN 5-LIKE
BC019337	IGHG1	IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 1
		(G1M MARKER)
BC056673	PPP1R2P9	PROTEIN PHOSPHATASE 1, REGULATORY
		(INHIBITOR) SUBUNIT 2 PSEUDOGENE 9
BC054520	MEF2D	MADS BOX TRANSCRIPTION ENHANCER FACTOR 2,
		POLYPEPTIDE D (MYOCYTE ENHANCER FACTOR
		2D)
NM_006902	PRRX1	PAIRED RELATED HOMEOBOX 1
NM_004436	ENSA	ENDOSULFINE ALPHA
NM_006255	PRKCH	PROTEIN KINASE C, ETA
NM_007080	LSM6	LSM6 HOMOLOG, U6 SMALL NUCLEAR RNA
		ASSOCIATED (S. CEREVISIAE)
NM_000860	HPGD	HYDROXYPROSTAGLANDIN DEHYDROGENASE 15-
		(NAD)
NM_144679	C17ORF56	CHROMOSOME 17 OPEN READING FRAME 56
NM_017431	PRKAG3	PROTEIN KINASE, AMP-ACTIVATED, GAMMA 3
		NON-CATALYTIC SUBUNIT
NM_031473	IFT81	INTRAFLAGELLAR TRANSPORT 81 HOMOLOG
		(CHLAMYDOMONAS)
BC064593	DCP2	DCP2 DECAPPING ENZYME HOMOLOG (S. CEREVISIAE)
BC007347	CHD2	CHROMODOMAIN HELICASE DNA BINDING
		PROTEIN 2
BC003690	IPO4	IMPORTIN 4
BC016327	NUP62CL	HYPOTHETICAL PROTEIN FLJ20130
NM_080600	MAG	MYELIN ASSOCIATED GLYCOPROTEIN
BC017258	MCM2	MCM2 MINICHROMOSOME MAINTENANCE
		DEFICIENT 2, MITOTIN (S. CEREVISIAE)
NM_017785	CCDC99	HYPOTHETICAL PROTEIN FLJ20364
BC000809	TCEAL1	TRANSCRIPTION ELONGATION FACTOR A (SII)-LIKE 1
NM_000485	APRT	ADENINE PHOSPHORIBOSYLTRANSFERASE
NM_138820	HIGD2A	HIG1 DOMAIN FAMILY, MEMBER 2A
BC009415	KIF26A	KINESIN FAMILY MEMBER 26A
BC017440	TRAPPC2L	HEMATOPOIETIC STEM/PROGENITOR CELLS 176
NM_001092	ABR	ACTIVE BCR-RELATED GENE
BC013352	HTF9C	HPAII TINY FRAGMENTS LOCUS 9C
NM_021947	SRR	SERINE RACEMASE
BC011585	PRKCDBP	PROTEIN KINASE C, DELTA BINDING PROTEIN
BC052600	ZNF718	ZINC FINGER PROTEIN 718
BC004518	SYT17	SYNAPTOTAGMIN XVII
NM_178509	STXBP4	SYNTAXIN BINDING PROTEIN 4
BC017770	NA	NA
BC066938	DDX43	DEAD (ASP-GLU-ALA-ASP) BOX POLYPEPTIDE 43
BC000393	FAM127B	DKFZP564B147 PROTEIN
BC025787	ALKBH1	ALKB, ALKYLATION REPAIR HOMOLOG 1 (E. COLI)
BC015944	TIA1	TIA1 CYTOTOXIC GRANULE-ASSOCIATED RNA
		BINDING PROTEIN
NM_017988	SCYL2	SCY1-LIKE 2 (S. CEREVISIAE)
NM_002020	FLT4	FMS-RELATED TYROSINE KINASE 4
NM_031472	TRPT1	TRNA PHOSPHOTRANSFERASE 1
BC001728*	TFPT	TCF3 (E2A) FUSION PARTNER (IN CHILDHOOD
		LEUKEMIA)
BC003566*	ZNF24	ZINC FINGER PROTEIN 24 (KOX 17)
BC005383*	CETN3	CENTRIN, EF-HAND PROTEIN, 3 (CDC31 HOMOLOG,
		YEAST)
BC007048*	ZMYM5	ZINC FINGER, MYM-TYPE 5
BC010125*	C3ORF37	CHROMOSOME 3 OPEN READING FRAME 37
BC011804*	C1ORF165	CHROMOSOME 1 OPEN READING FRAME 165
BC015803*	IRF2	INTERFERON REGULATORY FACTOR 2
BC017314*	ETS1	V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENE
		HOMOLOG 1 (AVIAN)
BC036335*	BTBD12	BTB (POZ) DOMAIN CONTAINING 12
BC036572*	ZCCHC12	ZINC FINGER, CCHC DOMAIN CONTAINING 12
BC051688*	FLJ10781	HYPOTHETICAL PROTEIN FLJ10781
BC056402*	LOC144097	HYPOTHETICAL PROTEIN BC007540
BC067299*	MDM4	MDM4, TRANSFORMED 3T3 CELL DOUBLE MINUTE
		4, P53 BINDING PROTEIN (MOUSE)
NM_000176*	NR3C1	NUCLEAR RECEPTOR SUBFAMILY 3, GROUP C,
		MEMBER 1 (GLUCOCORTICOID RECEPTOR)
NM_001008239*	C18ORF25	CHROMOSOME 18 OPEN READING FRAME 25
NM_001722*	POLR3D	POLYMERASE (RNA) III (DNA DIRECTED)
		POLYPEPTIDE D, 44 KDA
NM_001895*	CSNK2A1	CASEIN KINASE 2, ALPHA 1 POLYPEPTIDE
NM_002739*	PRKCG	PROTEIN KINASE C, GAMMA
NM_002938*	RNF4	RING FINGER PROTEIN 4
NM_003141*	TRIM21	TRIPARTITE MOTIF-CONTAINING 21
NM_003345*	UBE2I	UBIQUITIN-CONJUGATING ENZYME E2I (UBC9
		HOMOLOG, YEAST)
NM_003352*	SUMO1	SMT3 SUPPRESSOR OF MIF TWO 3 HOMOLOG 1
		(YEAST)
NM_004454*	ETV5	ETS VARIANT GENE 5 (ETS-RELATED MOLECULE)
NM_006977*	ZBTB25	ZINC FINGER AND BTB DOMAIN CONTAINING 25
NM_014720*	SLK	STE20-LIKE KINASE (YEAST)
NM_032141*	CCDC55	COILED-COIL DOMAIN CONTAINING 55
NM_145796*	POGZ	POGO TRANSPOSABLE ELEMENT WITH ZNF
		DOMAIN
NM_175907*	ZADH2	HYPOTHETICAL PROTEIN BC010734
NM_212540*	E2F6	E2F TRANSCRIPTION FACTOR 6

UFM1

NM_005879	TRAIP	TRAF INTERACTING PROTEIN
NM_001018	RPS15	RIBOSOMAL PROTEIN S15
NM_013974	DDAH2	DIMETHYLARGININE
		DIMETHYLAMINOHYDROLASE 2
NM_001278	CHUK	CONSERVED HELIX-LOOP-HELIX UBIQUITOUS
		KINASE
BC012611	EIF4E	EUKARYOTIC TRANSLATION INITIATION FACTOR
		4E
NM_006819	STIP1	STRESS-INDUCED-PHOSPHOPROTEIN 1
		(HSP70/HSP90-ORGANIZING PROTEIN)
NM_024647	NUP43	NUCLEOPORIN 43 KDA
NM_007045	FGFR1OP	FGFR1 ONCOGENE PARTNER
NM_014460	CSDC2	COLD SHOCK DOMAIN CONTAINING C2, RNA
		BINDING
NM_021260	ZFYVE1	ZINC FINGER, FYVE DOMAIN CONTAINING 1
NM_017437	CPSF2	CLEAVAGE AND POLYADENYLATION SPECIFIC
		FACTOR 2, 100 KDA
NM_138722	BCL2L14	BCL2-LIKE 14 (APOPTOSIS FACILITATOR)
NM_016059	PPIL1	PEPTIDYLPROLYL ISOMERASE (CYCLOPHILIN)-LIKE 1
NM_020139	BDH2	3-HYDROXYBUTYRATE DEHYDROGENASE, TYPE 2
NM_182493	MLCK	MLCK PROTEIN
BC000578	HPRT1	HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE 1
		(LESCH-NYHAN SYNDROME)
BC060785	TRIM40	TRIPARTITE MOTIF-CONTAINING 40
BC003132	NUDC	NUCLEAR DISTRIBUTION GENE C HOMOLOG (A. NIDULANS)
NM_031219	HDHD3	HALOACID DEHALOGENASE-LIKE HYDROLASE
		DOMAIN CONTAINING 3
NM_002358	MAD2L1	MAD2 MITOTIC ARREST DEFICIENT-LIKE 1 (YEAST)
NM_006578	GNB5	GUANINE NUCLEOTIDE BINDING PROTEIN (G
		PROTEIN), BETA 5
NM_004064	CDKN1B	CYCLIN-DEPENDENT KINASE INHIBITOR 1B (P27,
		KIP1)
BC030280	KIAA0513	KIAA0513
NM_005338	HIP1	HUNTINGTIN INTERACTING PROTEIN 1
NM_004881	TP53I3	TUMOR PROTEIN P53 INDUCIBLE PROTEIN 3
BC015395	CCDC148	HYPOTHETICAL PROTEIN BC015395
NM_000394	CRYAA	CRYSTALLIN, ALPHA A
BC005955	C8ORF53	CHROMOSOME 8 OPEN READING FRAME 53
BC001327	IFRD2	INTERFERON-RELATED DEVELOPMENTAL
		REGULATOR 2
BC021551	NFATC2IP	NUCLEAR FACTOR OF ACTIVATED T-CELLS,
		CYTOPLASMIC, CALCINEURIN-DEPENDENT 2
		INTERACTING PROTEIN
BC050537	FLJ20160	FLJ20160 PROTEIN
BC058862	TSKS	TESTIS-SPECIFIC KINASE SUBSTRATE
NM_005235	ERBB4	V-ERB-A ERYTHROBLASTIC LEUKEMIA VIRAL
		ONCOGENE HOMOLOG 4 (AVIAN)
NM_014012	REM1	RAS (RAD AND GEM)-LIKE GTP-BINDING 1
NM_022110	FKBPL	FK506 BINDING PROTEIN LIKE
NM_006147	IRF6	INTERFERON REGULATORY FACTOR 6
NM_001349	DARS	ASPARTYL-TRNA SYNTHETASE
BC064945	SCYL1BP1	SCY1-LIKE 1 BINDING PROTEIN 1
NM_032385	C5ORF4	CHROMOSOME 5 OPEN READING FRAME 4
NM_172037	RDH10	RETINOL DEHYDROGENASE 10 (ALL-TRANS)
NM_173621	C17ORF44	CHROMOSOME 17 OPEN READING FRAME 44
NM_004074	COX8A	CYTOCHROME C OXIDASE SUBUNIT 8A
		(UBIQUITOUS)
NM_022156	DUS1L	DIHYDROURIDINE SYNTHASE 1-LIKE (S. CEREVISIAE)
NM_016401	C11ORF73	HYPOTHETICAL PROTEIN HSPC138
NM_019617	GKN1	GASTROKINE 1
BC054501	DNM2	DYNAMIN 2
NM_058173	MUCL1	SMALL BREAST EPITHELIAL MUCIN
BC032307	CCDC123	HYPOTHETICAL PROTEIN FLJ14640
BC034028	SHARPIN	SHANK-ASSOCIATED RH DOMAIN INTERACTOR
BC015202	CENPT	CHROMOSOME 16 OPEN READING FRAME 56
BC013957	FAM62B	FAMILY WITH SEQUENCE SIMILARITY 62 (C2
		DOMAIN CONTAINING) MEMBER B
BC015569	ARL6IP4	ADP-RIBOSYLATION-LIKE FACTOR 6 INTERACTING
		PROTEIN 4
BC020221	STAC	SH3 AND CYSTEINE RICH DOMAIN
BC053895	IRS1	INSULIN RECEPTOR SUBSTRATE 1
NM_002748	MAPK6	MITOGEN-ACTIVATED PROTEIN KINASE 6
NM_198086	JUB	JUB, AJUBA HOMOLOG (XENOPUS LAEVIS)
NM_006621	AHCYL1	S-ADENOSYLHOMOCYSTEINE HYDROLASE-LIKE 1
NM_018698	NXT2	NUCLEAR TRANSPORT FACTOR 2-LIKE EXPORT
		FACTOR 2
NM_005034	POLR2K	POLYMERASE (RNA) II (DNA DIRECTED)
		POLYPEPTIDE K, 7.0 KDA
NM_018438	FBXO6	F-BOX PROTEIN 6
NM_033547	INTS4	INTEGRATOR COMPLEX SUBUNIT 4
NM_153212	GJB4	GAP JUNCTION PROTEIN, BETA 4 (CONNEXIN 30.3)
NM_175738	RAB37	RAB37, MEMBER RAS ONCOGENE FAMILY
BC013031	PHLDB1	PLECKSTRIN HOMOLOGY-LIKE DOMAIN, FAMILY B,
		MEMBER 1
NM_001005465	OR10G3	OLFACTORY RECEPTOR, FAMILY 10, SUBFAMILY G,
		MEMBER 3
NM_001899	CST4	CYSTATIN S
NM_004753	DHRS3	DEHYDROGENASE/REDUCTASE (SDR FAMILY)
		MEMBER 3
NM_021992	TMSL8	THYMOSIN-LIKE 8
NM_197970	BOLL	BOL, BOULE-LIKE (DROSOPHILA)
NM_139246	C9ORF97	CHROMOSOME 9 OPEN READING FRAME 97
NM_005586	MDFI	MYOD FAMILY INHIBITOR
BC041831	TLE3	TRANSDUCIN-LIKE ENHANCER OF SPLIT 3 (E(SP1)
		HOMOLOG, DROSOPHILA)
NM_003130	SRI	SORCIN
BC030237	SLC22A18AS	SOLUTE CARRIER FAMILY 22 (ORGANIC CATION
		TRANSPORTER), MEMBER 18 ANTISENSE
BC053351	DLX1	DISTAL-LESS HOMEOBOX 1
BC022034	LDHAL6B	LACTATE DEHYDROGENASE A-LIKE 6B
BC031964	GLUL	GLUTAMATE-AMMONIA LIGASE (GLUTAMINE
		SYNTHETASE)
NM_032350	C7ORF50	HYPOTHETICAL PROTEIN MGC11257
NM_152646		hypothetical protein MGC23270
BC024245	SALL2	SAL-LIKE 2 (DROSOPHILA)
NM_001004300	ZNF720	ZINC FINGER PROTEIN 720
NM_079422	MYL1	MYOSIN, LIGHT POLYPEPTIDE 1, ALKALI;
		SKELETAL, FAST
NM_024295	DERL1	DER1-LIKE DOMAIN FAMILY, MEMBER 1
BC026241	UBE3C	UBIQUITIN PROTEIN LIGASE E3C
BC064144	NA	NA
NM_152266	C19ORF40	HYPOTHETICAL PROTEIN MGC32020
NM_017722	TRMT1	TRM1 TRNA METHYLTRANSFERASE 1 HOMOLOG (S. CEREVISIAE)
NM_000905	NPY	NEUROPEPTIDE Y
BC001553	CHMP2B	CHROMATIN MODIFYING PROTEIN 2B
NM_006438	COLEC10	COLLECTIN SUB-FAMILY MEMBER 10 (C-TYPE
		LECTIN)
NM_014424	HSPB7	HEAT SHOCK 27 KDA PROTEIN FAMILY, MEMBER 7
		(CARDIOVASCULAR)
NM_001179	ART3	ADP-RIBOSYLTRANSFERASE 3
NM_020348	CNNM1	CYCLIN M1
NM_006928	SILV	SILVER HOMOLOG (MOUSE)
NM_022568	ALDH8A1	ALDEHYDE DEHYDROGENASE 8 FAMILY, MEMBER
		A1
NM_178152	DCX	DOUBLECORTEX; LISSENCEPHALY, X-LINKED
		(DOUBLECORTIN)
NM_153822	PSMD4	PROTEASOME (PROSOME, MACROPAIN) 26S
		SUBUNIT, NON-ATPASE, 4
NM_001699	AXL	AXL RECEPTOR TYROSINE KINASE
BC006195	ACLY	ATP CITRATE LYASE
NM_020397	CAMK1D	CALCIUM/CALMODULIN-DEPENDENT PROTEIN
		KINASE ID
BC017249	ENO3	ENOLASE 1, (ALPHA)
BC001600	CDC123	CHROMOSOME 10 OPEN READING FRAME 7
NM_024770	METTL8	HYPOTHETICAL PROTEIN FLJ13984
NM_194270	MORN2	MORN REPEAT CONTAINING 2
NM_022650	RASA1	RAS P21 PROTEIN ACTIVATOR (GTPASE
		ACTIVATING PROTEIN) 1
BC005830	ANXA9	ANNEXIN A9
NM_014065	ASTE1	ASTEROID HOMOLOG 1 (DROSOPHILA)
BC014244	RTN2	RETICULON 2
BC024002	FNDC8	FIBRONECTIN TYPE III DOMAIN CONTAINING 8
NM_178034	PLA2G4D	PHOSPHOLIPASE A2, GROUP IVD (CYTOSOLIC)
BC025266	TASP1	TASPASE, THREONINE ASPARTASE, 1
NM_003928	FAM127A	CAAX BOX 1
NM_017819	LOC131909	RNA (GUANINE-9-) METHYLTRANSFERASE DOMAIN
		CONTAINING 1
NM_018158	SLC4A1AP	SOLUTE CARRIER FAMILY 4 (ANION EXCHANGER),
		MEMBER 1, ADAPTOR PROTEIN
NM_175571	GIMAP8	GTPASE, IMAP FAMILY MEMBER 8
BC000453	PCM1	PERICENTRIOLAR MATERIAL 1
NM_000910	NPY2R	NEUROPEPTIDE Y RECEPTOR Y2
NM_018679	TCP11	T-COMPLEX 11 (MOUSE)
NM_022559	GH1	CHORIONIC SOMATOMAMMOTROPIN HORMONE 1
		(PLACENTAL LACTOGEN)
BC030957	ANK1	ANKYRIN 1, ERYTHROCYTIC
NM_003168	SUPT4H1	SUPPRESSOR OF TY 4 HOMOLOG 1 (S. CEREVISIAE)
BC012095	BST1	BONE MARROW STROMAL CELL ANTIGEN 1
BC013740	SLC2A6	SOLUTE CARRIER FAMILY 2 (FACILITATED
		GLUCOSE TRANSPORTER), MEMBER 6
NM_016505	ZCCHC17	ZINC FINGER, CCHC DOMAIN CONTAINING 17
NM_018697	LANCL2	LANC LANTIBIOTIC SYNTHETASE COMPONENT C-
		LIKE 2 (BACTERIAL)
NM_152619	DCLK2	DOUBLECORTIN AND CAM KINASE-LIKE 2
NM_152770	C4ORF22	HYPOTHETICAL PROTEIN MGC35043
NM_004401	DFFA	DNA FRAGMENTATION FACTOR, 45 KDA, ALPHA
		POLYPEPTIDE
NM_030636	EEPD1	KIAA1706 PROTEIN
BC014260	PARP3	POLY (ADP-RIBOSE) POLYMERASE FAMILY,
		MEMBER 3
BC009010	C6ORF142	CHROMOSOME 6 OPEN READING FRAME 142
BC047722	C2ORF64	HYPOTHETICAL PROTEIN MGC52110
NM_080873	ASB11	ANKYRIN REPEAT AND SOCS BOX-CONTAINING 11
NM_173547	TRIM65	TRIPARTITE MOTIF-CONTAINING 65
BC041668	RIPK3	RECEPTOR-INTERACTING SERINE-THREONINE
		KINASE 3
BC033728	NA	NA
BC048217	SPATA5	SPERMATOGENESIS ASSOCIATED 5
NM_001001852	PIM3	PIM-3 ONCOGENE
NM_002904	RDBP	RD RNA BINDING PROTEIN
BC030608	PODN	PODOCAN
BC023982	C5ORF32	PUTATIVE NUCLEAR PROTEIN ORF1-FL49
NM_133332	WHSC1	WOLF-HIRSCHHORN SYNDROME CANDIDATE 1
NM_004040	RHOB	RAS HOMOLOG GENE FAMILY, MEMBER B
BC033708	RALGPS1	RAL GEF WITH PH DOMAIN AND SH3 BINDING
		MOTIF 1
NM_002491	NDUFB3	NADH DEHYDROGENASE (UBIQUINONE) 1 BETA
		SUBCOMPLEX, 3, 12 KDA
BC015944	TIA1	TIA1 CYTOTOXIC GRANULE-ASSOCIATED RNA
		BINDING PROTEIN
BC050688	RPSA	RIBOSOMAL PROTEIN SA
NM_002443	MSMB	MICROSEMINOPROTEIN, BETA-
NM_172314	IL25	INTERLEUKIN 17E
NM_019845	RPRM	REPRIMO, TP53 DEPENDENT G2 ARREST MEDIATOR
		CANDIDATE
BC013163	DCUN1D1	DCN1, DEFECTIVE IN CULLIN NEDDYLATION 1,
		DOMAIN CONTAINING 1 (S. CEREVISIAE)
BC017741	GTDC1	PRO0159 PROTEIN
BC023152	GYG2	GLYCOGENIN 2
NM_005663	WHSC2	WOLF-HIRSCHHORN SYNDROME CANDIDATE 2
NM_000214	JAG1	JAGGED 1 (ALAGILLE SYNDROME)
NM_004403	DFNA5	DEAFNESS, AUTOSOMAL DOMINANT 5
NM_022073	EGLN3	HYPOTHETICAL PROTEIN FLJ21620
NM_030571	NDFIP1	NEDD4 FAMILY INTERACTING PROTEIN 1
NM_145252	LOC124220	SIMILAR TO COMMON SALIVARY PROTEIN 1
BC000772	SIPA1L3	SIGNAL-INDUCED PROLIFERATION-ASSOCIATED 1
		LIKE 3
NM_006579	EBP	EMOPAMIL BINDING PROTEIN (STEROL
		ISOMERASE)
BC014441	NSUN4	NOL1/NOP2/SUN DOMAIN FAMILY, MEMBER 4
BC019902	CCDC21	COILED-COIL DOMAIN CONTAINING 21
BC036827	LILRB2	LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTOR,
		SUBFAMILY B (WITH TM AND ITIM DOMAINS),
		MEMBER 2
NM_001680	FXYD2	FXYD DOMAIN CONTAINING ION TRANSPORT
		REGULATOR 2
NM_006439	MAB21L2	MAB-21-LIKE 2 (C. ELEGANS)
NM_032786	ZC3H10	ZINC FINGER CCCH-TYPE CONTAINING 10
NM_024613	PLEKHF2	PLECKSTRIN HOMOLOGY DOMAIN CONTAINING,
		FAMILY F (WITH FYVE DOMAIN) MEMBER 2
NM_001752	CAT	CATALASE
NM_152471		hypothetical protein MGC17515
NM_152716	PATL1	FLJ36874 PROTEIN
BC004243	BCAT2	BRANCHED CHAIN AMINOTRANSFERASE 2,
		MITOCHONDRIAL
BC056246	GALNT3	UDP-N-ACETYL-ALPHA-D-
		GALACTOSAMINE:POLYPEPTIDE N-
		ACETYLGALACTOSAMINYLTRANSFERASE 3
		(GALNAC-T3)
NM_022133	SNX16	SORTING NEXIN 16
NM_025221	KCNIP4	KV CHANNEL INTERACTING PROTEIN 4
NM_025234	WDR61	WD REPEAT DOMAIN 61
BC014649	GAL3ST1	GALACTOSE-3-O-SULFOTRANSFERASE 1
NM_002734	PRKAR1A	PROTEIN KINASE, CAMP-DEPENDENT,
		REGULATORY, TYPE I, ALPHA (TISSUE SPECIFIC
		EXTINGUISHER 1)
NM_023934	FUNDC2	FUN14 DOMAIN CONTAINING 2
NM_145173	DIRAS1	DIRAS FAMILY, GTP-BINDING RAS-LIKE 1
NM_020142	NDUFA4L2	NADH:UBIQUINONE OXIDOREDUCTASE MLRQ
		SUBUNIT HOMOLOG
NM_016485	VTA1	CHROMOSOME 6 OPEN READING FRAME 55
NM_000345	SNCA	SYNUCLEIN, ALPHA (NON A4 COMPONENT OF
		AMYLOID PRECURSOR)
BC067447	DAB1	DISABLED HOMOLOG 1 (DROSOPHILA)
NM_001010971	SAMD13	STERILE ALPHA MOTIF DOMAIN CONTAINING 13
BC022043	C7ORF36	CHROMOSOME 7 OPEN READING FRAME 36
BC004233*	TTYH2	TWEETY HOMOLOG 2 (DROSOPHILA)
BC017504*	DEF6	DIFFERENTIALLY EXPRESSED IN FDCP 6 HOMOLOG
		(MOUSE)
BC018206*	FAM128B	HYPOTHETICAL PROTEIN FLJ14346
BC018404*	FGF21	FIBROBLAST GROWTH FACTOR 21
BC020985*	COASY	COENZYME A SYNTHASE
BC031469*	LOC554207	HYPOTHETICAL LOC554207
BC058924*	UBE2M	UBIQUITIN-CONJUGATING ENZYME E2M (UBC12
		HOMOLOG, YEAST)
NM_000020*	ACVRL1	ACTIVIN A RECEPTOR TYPE II-LIKE 1
NM_000154*	GALK1	GALACTOKINASE 1
NM_001014796*	DDR2	DISCOIDIN DOMAIN RECEPTOR FAMILY, MEMBER 2
NM_001105*	ACVR1	ACTIVIN A RECEPTOR, TYPE I
NM_001752*	CAT	CATALASE
NM_002227*	JAK1	JANUS KINASE 1 (A PROTEIN TYROSINE KINASE)
NM_002498*	NEK3	NIMA (NEVER IN MITOSIS GENE A)-RELATED
		KINASE 3
NM_002964*	S100A8	S100 CALCIUM BINDING PROTEIN A8
		(CALGRANULIN A)
NM_003063*	SLN	SARCOLIPIN
NM_004972*	JAK2	JANUS KINASE 2 (A PROTEIN TYROSINE KINASE)
NM_005036*	PPARA	PEROXISOME PROLIFERATIVE ACTIVATED
		RECEPTOR, ALPHA
NM_005122*	NR1I3	NUCLEAR RECEPTOR SUBFAMILY 1, GROUP I,
		MEMBER 3
NM_005123*	NR1H4	NUCLEAR RECEPTOR SUBFAMILY 1, GROUP H,
		MEMBER 4
NM_014583*	LMCD1	LIM AND CYSTEINE-RICH DOMAINS 1
NM_015646*	RAP1B	RAP1B, MEMBER OF RAS ONCOGENE FAMILY
NM_016495*	TBC1D7	TBC1 DOMAIN FAMILY, MEMBER 7
NM_021709*	SIVA1	CD27-BINDING (SIVA) PROTEIN
NM_030572*	C12ORF39	CHROMOSOME 12 OPEN READING FRAME 39
NM_033360*	KRAS	V-HA-RAS HARVEY RAT SARCOMA VIRAL
		ONCOGENE HOMOLOG
NM_130807*	MOBKL2A	MOB1, MPS ONE BINDER KINASE ACTIVATOR-LIKE
		2A (YEAST)
NM_145173*	DIRAS1	DIRAS FAMILY, GTP-BINDING RAS-LIKE 1
NM_173541*	C10ORF91	CHROMOSOME 10 OPEN READING FRAME 91
BC004233*	NA	NA
BC008624*	NA	NA

ISG15

BC013366*	URP2	UNC-112 RELATED PROTEIN 2
BC017314*	ETS1	V-ETS ERYTHROBLASTOSIS VIRUS E26 ONCOGENE
		HOMOLOG 1 (AVIAN)
BC018404*	FGF21	FIBROBLAST GROWTH FACTOR 21
BC022363*	VPS37A	VACUOLAR PROTEIN SORTING 37A (YEAST)
BC024725*	ANKRD50	ANKYRIN REPEAT DOMAIN 50
BC025307*	PRKD2	PROTEIN KINASE D2
BC029112*	SAMSN1	SAM DOMAIN, SH3 DOMAIN AND NUCLEAR
		LOCALISATION SIGNALS, 1
BC029480*	LOC554203	HYPOTHETICAL LOC554203
BC035636*	APBB1IP	AMYLOID BETA (A4) PRECURSOR PROTEIN-
		BINDING, FAMILY B, MEMBER 1 INTERACTING
		PROTEIN
BC038838*	PRR16	MESENCHYMAL STEM CELL PROTEIN DSC54
BC039244*	NFYA	NUCLEAR TRANSCRIPTION FACTOR Y, ALPHA
BC042999*	ASXL2	ADDITIONAL SEX COMBS LIKE 2 (DROSOPHILA)
BC062423*	C7ORF41	HYPOTHETICAL PROTEIN ELLS1
NM_001571*	IRF3	INTERFERON REGULATORY FACTOR 3
NM_001926*	DEFA6	DEFENSIN, ALPHA 6, PANETH CELL-SPECIFIC
NM_002505*	NFYA	NUCLEAR TRANSCRIPTION FACTOR Y, ALPHA
NM_003141*	TRIM21	TRIPARTITE MOTIF-CONTAINING 21
NM_004304*	ALK	ANAPLASTIC LYMPHOMA KINASE (KI-1)
NM_005214*	CTLA4	CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED
		PROTEIN 4
NM_005902*	SMAD3	SMAD, MOTHERS AGAINST DPP HOMOLOG 3
		(DROSOPHILA)
NM_006324*	CFDP1	CRANIOFACIAL DEVELOPMENT PROTEIN 1
NM_007242*	DDX19B	DEAD (ASP-GLU-ALA-AS) BOX POLYPEPTIDE 19B
NM_012472*	LRRC6	LEUCINE RICH REPEAT CONTAINING 6
NM_015927*	TGFB1I1	TRANSFORMING GROWTH FACTOR BETA 1
		INDUCED TRANSCRIPT 1
NM_017724*	LRRFIP2	LEUCINE RICH REPEAT (IN FLII) INTERACTING
		PROTEIN 2
NM_017855*	ODAM	APIN PROTEIN
NM_023112*	OTUB2	OTU DOMAIN, UBIQUITIN ALDEHYDE BINDING 2
NM_025241*	UBXD1	UBX DOMAIN CONTAINING 1
NM_053283*	DCD	DERMCIDIN
NM_172160*	KCNAB1	POTASSIUM VOLTAGE-GATED CHANNEL, SHAKER-
		RELATED SUBFAMILY, BETA MEMBER 1
NM_175907*	ZADH2	HYPOTHETICAL PROTEIN BC010734

The protein targets showing the highest reactivity in a sumol PTM assay were the RANBP2 protein, which was previously identified as a sumol 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.
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, sumol, 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

- 1. M. Rape, S. K. Reddy, M. W. Kirschner, Cell 124, 89 (Jan. 13, 2006).
- 2. N. G. Ayad, S. Rankin, D. Ooi, M. Rape, M. W. Kirschner, Methods Enzymol 399, 404 (2005).
- 3. T. J. McGarry, M. W. Kirschner, Cell 93, 1043 (Jun. 12, 1998).
- 4. R. W. King, K. D. Lustig, P. T. Stukenberg, T. J. McGarry, M. W. Kirschner, Science 277, 973 (Aug. 15, 1997).
- 5. K. D. Lustig et al., Methods Enzymol 283, 83 (1997).
- 6. F. Kanai et al., Embo J 19, 6778 (Dec. 15, 2000).
- 7. Y. H. Oh et al., Biosens Bioelectron 22, 1260 (Feb. 15, 2007).
- 8. R. Gupta et al., Mol Syst Biol 3, 116 (2007).
- 9. J. Ptacek et al., Nature 438, 679 (Dec. 1, 2005).
- 10. C. Schnack, K. M. Danzer, B. Hengerer, F. Gillardon, Neuroscience (Feb. 29, 2008).
- 11. T. Evans, E. T. Rosenthal, J. Youngblom, D. Distel, T. Hunt, Cell 33, 389 (June 1983).
- 12. M. Glotzer, A. W. Murray, M. W. Kirschner, Nature 349, 132 (Jan. 10, 1991).
- 13. R. W. King et al., Cell 81, 279 (Apr. 21, 1995).
- 14. G. Fang, H. Yu, M. W. Kirschner, Philos Trans R Soc Lond B Biol Sci 354, 1583 (Sep. 29, 1999).
- 15. R. W. King, R. J. Deshaies, J. M. Peters, M. W. Kirschner, Science 274, 1652 (Dec. 6, 1996).
- 16. H. Yu, R. W. King, J. M. Peters, M. W. Kirschner, Curr Biol 6, 455 (Apr. 1, 1996).
- 17. S. K. Reddy, M. Rape, W. A. Margansky, M. W. Kirschner, Nature 446, 921 (Apr. 19, 2007).
- 18. D. Verdick, S. Handran, and S. Pickett., p. 83-98. In G. Kamberova (ed.), DNA image analysis: nuts and bolts, DNA Press LLC, Salem, Mass., Key considerations for accurate microarray scanning and image analysis. K. G. (ed), Ed., DNA Array Image Analysis: Nuts and Bolts. (DNA Press LLC: Salem, Mass. 2002.), pp. 83-98.
- 19. J. G. Chafouleas, W. E. Bolton, H. idaka, A. E. Boyd, 3rd, A. R. eans, Cell 28, 41 (January 1982).
- 20. J. Roig, A. Mikhailov, C. Belham, J. Avruch, Genes Dev 16, 1640 (Jul. 1, 2002).
- 21. J. Roig, A. Groen, J. Caldwell, J. Avruch, Mol Biol Cell 16, 4827 (October 2005).
- 22. M. J. Kallio, V. A. Beardmore, J. Weinstein, G. J. Gorbsky, J Cell Biol 158, 841 (Sep. 2, 2002).
- 23. J. W. Raff, K. Jeffers, J. Y. Huang, J Cell Biol 157, 1139 (Jun. 24, 2002).
- 24. J. Huang, J. W. Raff, Embo J 18, 2184 (Apr. 15, 1999).
- 25. Y. A. Lam et al., Proc Natl Acad Sci USA 97, 9902 (Aug. 29, 2000).
- 26. K L. Lim, V. L. Dawson, T. M. Dawson, Neurobiol Aging 27, 524 (April 2006).
- 27. J. A. Olzmann, L. S. Chin, Autophagy 4, 85 (January-February 2008).
- 28. J. M. Tan et al., Hum Mol Genet 17, 431 (Feb. 1, 2008).
- 29. S. Adhikary et al., Cell 123, 409 (Nov. 4, 2005).
- 30. C. L. Brooks, W. Gu, Cell Cycle 3, 895 (July 2004).
- 31. A. D. Choudhury, H. Xu, R. Baer, J Biol Chem 279, 33909 (Aug. 6, 2004).
- 32. B. M. Bolstad, R. A. Irizarry, M. Astrand, T. P. Speed, Bioinformatics 19, 185 (Jan. 22, 2003).
- 33. Adhikary, S., Marinoni, F., Hock, A., Hulleman, E., Popov, N., Beier, R., Bernard, S., Quarto, M., Capra, M., Goettig, S., et al. 2005. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation. Cell 123:409-421.
- 34. Choudhury, A. D., Xu, H., and Baer, R. 2004. Ubiquitination and proteasomal degradation of the BRCA1 tumor suppressor is regulated during cell cycle progression. J Biol Chem 279:33909-33918.
- 35. Mo, Y. Y., Yu, Y., Theodosiou, E., Rachel Ee, P. L., and Beck, W. T. 2005. A role for Ubc9 in tumorigenesis. Oncogene 24:2677-2683.
- 36. Lam, Y. A., Pickart, C. M., Alban, A., Landon, M., Jamieson, C., Ramage, R., Mayer, R. J., and Layfield, R. 2000. Inhibition of the ubiquitin-proteasome system in Alzheimer's disease. Proc Natl Acad Sci USA 97:9902-9906.
- 37. Wang, J. Z., Grundke-Iqbal, I., and Iqbal, K. 1996. Glycosylation of microtubule-associated protein tau: an abnormal posttranslational modification in Alzheimer's disease. Nat Med 2:871-875.
- 38. Lim, K. L., Dawson, V. L., and Dawson, T. M. 2006. Parkin-mediated lysine 63-linked polyubiquitination: a link to protein inclusions formation in Parkinson's and other conformational diseases? Neurobiol Aging 27:524-529.
- 39. Olzmann, J. A., and Chin, L. S. 2008. Parkin-mediated K63-linked polyubiquitination: a signal for targeting misfolded proteins to the aggresome-autophagy pathway. Autophagy 4:85-87.
- 40. Tan, J. M., Wong, E. S., Kirkpatrick, D. S., Pletnikova, O., Ko, H. S., Tay, S. P., Ho, M. W., Troncoso, J., Gygi, S. P., Lee, M. K., et al. 2008. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum Mol Genet 17:431-439.
- 41. Abel, T., and Zukin, R. S. 2008. Epigenetic targets of HDAC inhibition in neurodegenerative and psychiatric disorders. Curr Opin Pharmacol 8:57-64.
- 42. Mori, F., Nishie, M., Piao, Y. S., Kito, K., Kamitani, T., Takahashi, H., and Wakabayashi, K. 2005. Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders. Neuropathol Appl Neurobiol 31:53-61.
- 43. Braunstein, I., Miniowitz, S., Moshe, Y., and Hershko, A. 2007. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint. Proc Natl Acad Sci USA 104:4870-4875.
- 44. Ciechanover, A., Finley, D., and Varshaysky, A. 1984. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37:57-66.
- 45. Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T. 1983. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33:389-396.
- 46. Fang, G., Yu, H., and Kirschner, M. W. 1999. Control of mitotic transitions by the anaphase-promoting complex. Philos Trans R Soc Lond B Biol Sci 354:1583-1590.
- 47. Glotzer, M., Murray, A. W., and Kirschner, M. W. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349:132-138.
- 48. King, R. W., Deshaies, R. J., Peters, J. M., and Kirschner, M. W. 1996. How proteolysis drives the cell cycle. Science 274:1652-1659.
- 49. Reddy, S. K., Rape, M., Margansky, W. A., and Kirschner, M. W. 2007. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446:921-925.
- 50. Dery, U., and Masson, J. Y. 2007. Twists and turns in the function of DNA damage signaling and repair proteins by PTMs. DNA Repair (Amst) 6:561-577.
- 51. Sadri-Vakili, G., and Cha, J. H. 2006. Mechanisms of disease: Histone modifications in Huntington's disease. Nat Clin Pract Neurol 2:330-338.
- 52. Steffan, J. S., Agrawal, N., Pallos, J., Rockabrand, E., Trotman, L. C., Slepko, N., Ines, K., Lukacsovich, T., Zhu, Y. Z., Cattaneo, E., et al. 2004. SUMO modification of Huntingtin and Huntington's disease pathology. Science 304:100-104.
- 53. Perkins, N. D. 2006. PTMs regulating the activity and function of the nuclear factor kappa B pathway. Oncogene 25:6717-6730.
- 54. Takahashi-Fujigasaki, J., Arai, K., Funata, N., and Fujigasaki, H. 2006. SUMOylation substrates in neuronal intranuclear inclusion disease. Neuropathol Appl Neurobiol 32:92-100.
- 55. Li, M., Guo, D., Isales, C. M., Eizirik, D. L., Atkinson, M., She, J. X., and Wang, C. Y. 2005. SUMO wrestling with type 1 diabetes. J Mol Med 83:504-513.
- 56. Ueda, H., Goto, J., Hashida, H., Lin, X., Oyanagi, K., Kawano, H., Zoghbi, H. Y., Kanazawa, I., and Okazawa, H. 2002. Enhanced SUMOylation in polyglutamine diseases. Biochem Biophys Res Commun 293:307-313.
- 57. Sufan, R. I., Jewett, M. A., and Ohh, M. 2004. The role of von Hippel-Lindau tumor suppressor protein and hypoxia in renal clear cell carcinoma. Am J Physiol Renal Physiol 287:F1-6.
- 58. Wada, H., Yeh, E. T., and Kamitani, T. 1999. The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2. J Biol Chem 274:36025-36029.
- 59. Shishido, T., Woo, C. H., Ding, B., McClain, C., Molina, C. A., Yan, C., Yang, J., and Abe, J. 2008. Effects of MEK5/ERK5 association on small ubiquitin-related modification of ERK5: implications for diabetic ventricular dysfunction after myocardial infarction. Circ Res 102:1416-1425.
- 60. Ciechanover, A., and Brundin, P. 2003. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40:427-446.
- 61. Kim, J. K., Mastronardi, F. G., Wood, D. D., Lubman, D. M., Zand, R., and Moscarello, M. A. 2003. Multiple sclerosis: an important role for PTMs of myelin basic protein in pathogenesis. Mol Cell Proteomics 2:453-462.
- 62. Matei, L. 1997. Plasma proteins glycosylation and its alteration in disease. Rom J Intern Med 35:3-11.
- 63. Shinbo, Y., Niki, T., Taira, T., Ooe, H., Takahashi-Niki, K., Maita, C., Seino, C., Iguchi-Ariga, S. M., and Ariga, H. 2006. Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities. Cell Death Differ 13:96-108.
- 64. Zhong, N., Kim, C. Y., Rizzu, P., Geula, C., Porter, D. R., Pothos, E. N., Squitieri, F., Heutink, P., and Xu, J. 2006. DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor. J Biol Chem 281:20940-20948.
- 65. Brooks, C. L., and Gu, W. 2003. Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 15:164-171.
- 66. Casalino, L., De Cesare, D., and Verde, P. 2003. Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization. Mol Cell Biol 23:4401-4415.
- 67. Fujioka, S., Sclabas, G. M., Schmidt, C., Niu, J., Frederick, W. A., Dong, Q. G., Abbruzzese, J. L., Evans, D. B., Baker, C., and Chiao, P. J. 2003. Inhibition of constitutive NF-kappa B activity by I kappa B alpha M suppresses tumorigenesis. Oncogene 22:1365-1370.
- 68. Lee, P. S., Chang, C., Liu, D., and Derynck, R. 2003. Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling. J Biol Chem 278:27853-27863.
- 69. Orii, A., Masutani, H., Nikaido, T., Zhai, Y. L., Kato, K., Kariya, M., Konishi, I., Yodoi, J., and Fujii, S. 2002. Altered PTM of redox factor 1 protein in human uterine smooth muscle tumors. J Clin Endocrinol Metab 87:3754-3759.
- 70. Turenne, G. A., and Price, B. D. 2001. Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity. BMC Cell Biol 2:12.
- 71. O'Connor, T. J., Neufeld, E., Bechberger, J., and Fujita, D. J. 1992. pp60c-src in human melanocytes and melanoma cells exhibits elevated specific activity and reduced tyrosine 530 phosphorylation compared to human fibroblast pp60c-src. Cell Growth Differ 3:435-442.
- 72. Reynolds, F. H., Jr., Van de Ven, W. J., and Stephenson, J. R. 1980. Abelson murine leukemia virus transformation-defective mutants with impaired P120-associated protein kinase activity. J Virol 36:374-386.
- 73. Haraguchi, T., Fisher, S., Olofsson, S., Endo, T., Groth, D., Tarentino, A., Borchelt, D. R., Teplow, D., Hood, L., Burlingame, A., et al. 1989. Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch Biochem Biophys 274:1-13.
- 74. Kalb, R., Neveling, K., Nanda, I., Schindler, D., and Hoehn, H. 2006. Fanconi anemia: causes and consequences of genetic instability. Genome Dyn 1:218-242.
- 75. Meetei, A. R., de Winter, J. P., Medhurst, A. L., Wallisch, M., Waisfisz, Q., van de Vrugt, H. J., Oostra, A. B., Yan, Z., Ling, C., Bishop, C. E., et al. 2003. A novel ubiquitin ligase is deficient in Fanconi anemia. Nat Genet 35:165-170.
- 76. Grillari, J., Katinger, H., and Voglauer, R. 2006. Aging and the ubiquitinome: traditional and non-traditional functions of ubiquitin in aging cells and tissues. Exp Gerontol 41:1067-1079.
- 77. Li, F., Zhang, L., Craddock, J., Bruce-Keller, A. J., Dasuri, K., Nguyen, A., and Keller, J. N. 2008. Aging and dietary restriction effects on ubiquitination, sumoylation, and the proteasome in the heart. Mech Ageing Dev 129:515-521.
- 78. Li, W., Gao, B., Lee, S. M., Bennett, K., and Fang, D. 2007. RLE-1, an E3 ubiquitin ligase, regulates C. elegans aging by catalyzing DAF-16 polyubiquitination. Dev Cell 12:235-246.
- 79. Zhang, L., Li, F., Dimayuga, E., Craddock, J., and Keller, J. N. 2007. Effects of aging and dietary restriction on ubiquitination, sumoylation, and the proteasome in the spleen. FEBS Lett 581:5543-5547.
- 80. Gaczynska, M., Osmulski, P. A., and Ward, W. F. 2001. Caretaker or undertaker? The role of the proteasome in aging. Mech Ageing Dev 122:235-254.
- 81. Yao, D., Gu, Z., Nakamura, T., Shi, Z. Q., Ma, Y., Gaston, B., Palmer, L. A., Rockenstein, E. M., Zhang, Z., Masliah, E., et al. 2004. Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity. Proc Natl Acad Sci USA 101:10810-10814.
- 82. Blough, H. A., Pauwels, R., De Clercq, E., Cogniaux, J., Sprecher-Goldberger, S., and Thiry, L. 1986. Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins. Biochem Biophys Res Commun 141:33-38.
- 83. Conrad, S. F., Byeon, I. J., DiGeorge, A. M., Lairmore, M. D., Tsai, M. D., and Kaumaya, P. T. 1995. Immunogenicity and conformational properties of an N-linked glycosylated peptide epitope of human T-lymphotropic virus type 1 (HTLV-1). Biomed Pept Proteins Nucleic Acids 1:83-92.
- 84. Lee, S., Shin, Y., Marler, J., and Levin, M. C. 2008. Post-translational glycosylation of target proteins implicate molecular mimicry in the pathogenesis of HTLV-1 associated neurological disease. J Neuroimmunol.
- 85. Schneider, J., Bayer, H., Bienzle, U., and Hunsmann, G. 1985. A glycopolypeptide (gp 100) is the main antigen detected by HTLV-III antisera. Med Microbiol Immunol 174:35-42.
- 86. Harauz, G., and Musse, A. A. 2007. A tale of two citrullines—structural and functional aspects of myelin basic protein deimination in health and disease. Neurochem Res 32:137-158.
- 87. Akimoto, Y., Hart, G. W., Hirano, H., and Kawakami, H. 2005. O-GlcNAc modification of nucleocytoplasmic proteins and diabetes. Med Mol Morphol 38:84-91.
- 88. Dias, W. B., and Hart, G. W. 2007. O-GlcNAc modification in diabetes and Alzheimer's disease. Mol Biosyst 3:766-772.
- 89. Jones, S. P. 2005. A bittersweet modification: O-GlcNAc and cardiac dysfunction. Circ Res 96:925-926.
- 90. Kudlow, J. E. 2006. PTM by O-GlcNAc: another way to change protein function. J Cell Biochem 98:1062-1075.
- 91. Doyle, H. A., and Mamula, M. J. 2002. Posttranslational protein modifications: new flavors in the menu of autoantigens. Curr Opin Rheumatol 14:244-249.
- 92. Doyle, H. A., and Mamula, M. J. 2005. Posttranslational modifications of self-antigens. Ann N Y Acad Sci 1050:1-9.
- 93. van Boekel, M. A., and van Venrooij, W. J. 2003. Modifications of arginines and their role in autoimmunity. Autoimmun Rev 2:57-62.
- 94. Zhou, Z., and Menard, H. A. 2002. Autoantigenic posttranslational modifications of proteins: does it apply to rheumatoid arthritis? Curr Opin Rheumatol 14:250-253.

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. 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.

2. The method of claim 1, further comprising 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.

3. The method of claim 1, wherein increase in the signal from the array compared to a background or the reaction with a control is indicative of increased PTM.

4. The method of claim 1, wherein decrease in the signal from the array compared to a background or the control is indicative of PTM alteration.

5. The method of claim 1, wherein 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.

6. The method of claim 5, wherein the antibody or antigen-binding fragment thereof, the natural or recombinant ligand, the small molecule, or the modifying moiety is labeled with a tag.

7. The method of claim 6, wherein the tag is a fluorescent molecule, a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a chemiluminescent moiety, magnetic particle, bioluminescent moiety, or peptide.

8. The method of claim 5, wherein the biochemical analysis is performed using mass spectroscopy, peptide mapping, or amino acid sequencing.

9. The method of claim 1, wherein the functional cell extract is not diluted prior to said contacting with the solid state array.

10. The method of claim 1, wherein the functional cell extract is obtained from a frozen or cryopreserved sample.

11. The method of claim 1, wherein an additional cellular energy source in the form of ATP is provided to the functional cell extract.

12. The method 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.

13. The method 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.

14. The method of claim 1, wherein the functional cell extract is derived from a specified cellular compartment.

15. The method of claim 14, wherein the cellular compartment is nucleus.

16. The method of claim 14, wherein the cellular compartment is cytosol.

17. The method of claim 14, wherein the cellular compartment is mitochondria.

18. The method of claim 1, wherein the functional cell extract is derived from a biological sample.

19. The method of claim 18, wherein the biological sample is selected from the group consisting of saliva, blood, serum, plasma, urine, cerebrospinal fluid, chorionic villus, placenta, solid tissue, amniotic fluid, a cell sample, and a tissue culture sample.

20. The method of claim 1, wherein 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.

21. The method of claim 1, wherein 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, deprenylation, demyristoylation, de-S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, deamidation, and any combination thereof.

22. The method 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.

23. The method of claim 1, wherein 2-10 PTM or PTM alterations thereof are identified simultaneously.