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

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

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US20100160177A1
US20100160177A1 US12/696,866 US69686610A US2010160177A1 US 20100160177 A1 US20100160177 A1 US 20100160177A1 US 69686610 A US69686610 A US 69686610A US 2010160177 A1 US2010160177 A1 US 2010160177A1
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ptm
protein
proteins
alteration
array
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Yifat Merbl
Marc W. Kirschner
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Harvard University
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Harvard University
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Assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE reassignment PRESIDENT AND FELLOWS OF HARVARD COLLEGE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KIRSCHNER, MARC W., MERBL, YIFAT
Publication of US20100160177A1 publication Critical patent/US20100160177A1/en
Priority to US14/796,408 priority patent/US11333668B2/en
Priority to US17/722,956 priority patent/US20220252612A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36—Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • PTM post-translational modification
  • the method further comprises identifying the effect of a test agent on the PTM or PTM alteration comprising the additional steps of:
  • an increase in the signal from the array compared to a background or the reaction with a control is indicative of increased PTM.
  • a decrease in the signal from the array compared to a background or the reaction with a control is indicative of PTM alteration.
  • 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.
  • the antibody or antigen-binding fragment thereof, the natural or recombinant ligand, the small molecule, or the modifying moiety is labeled with a tag.
  • the tag is a fluorescent molecule, a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a chemiluminescent moiety, magnetic particle, bioluminescent moiety, or peptide.
  • the biochemical analysis is performed using mass spectroscopy, peptide mapping, or amino acid sequencing.
  • 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.
  • the functional cell extract is obtained from a frozen or cryopreserved sample.
  • an additional cellular energy source in the form of ATP is provided to the functional cell extract.
  • the array comprising a plurality of proteins comprises at least one protein, protein fragment or peptide attached to the array without an added tag.
  • 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.
  • the functional cell extract is derived from a specified cellular compartment.
  • the cellular compartment is nucleus.
  • the cellular compartment is cytosol.
  • the cellular compartment is mitochondria.
  • the functional cell extract is derived from a biological sample.
  • 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.
  • 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.
  • 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.
  • DRB deubiquitination
  • dephosphorylation deglycosylation
  • desumoylation deacetylation
  • de-S-nitrosylation or denitrosylation decitrullination or dedeimination
  • deneddylation removal of OClcNAc
  • de-ADP-ribosylation demethylation
  • de-hydroxylation defattenylation
  • deufmylation deufmylation
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the standard produces a pattern of protein PTM that is diagnostic for a disease or medical condition, or the effects of a drug.
  • 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 35 S-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.
  • 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).
  • 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 35 S-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 35 S-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
  • 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.
  • CSF cerebrospinal fluid
  • 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.
  • 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.
  • DAB deubiquitination
  • dephosphorylation deglycosylation
  • desumoylation deacetylation
  • deS-nitrosylation or denitrosylation decitrullination or dedeimination
  • deneddylation removal of OClcNAc
  • 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.
  • 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.
  • 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.
  • 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.
  • PTM post-translational modification
  • the method further comprises identifying the effect of a test agent on the PTM or PTM alteration comprising the additional steps of:
  • 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.
  • 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.
  • agents are small molecules having a chemical moiety.
  • 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.
  • 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.
  • organic or inorganic compound e.g., including heterorganic and organometallic compounds
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modification moiety itself may be labeled directly.
  • 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.
  • antibodies may be labeled to assist in their direct detection.
  • 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.
  • 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-C
  • 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.
  • 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.
  • 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.
  • 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.
  • DRB deubiquitination
  • dephosphorylation deglycosylation
  • deacetylation deacetylation
  • de-S-nitrosylation or denitrosylation decitrullination or dedeimination
  • deneddylation removal of OClcNAc
  • de-ADP-ribosylation demethylation
  • 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.
  • DRB deubiquitination
  • 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.
  • DRB deubiquitination
  • dephosphorylation deglycosylation
  • deacetylation deacetylation
  • de-S-nitrosylation denitrosylation
  • decitrullination or dedeimination deneddylation
  • removal of OClcNAc de-ADP-ribosylation
  • demethylation
  • PTM post-translational modification
  • 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 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, ⁇ -bonding, ⁇ -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.
  • a chemical moiety also referred to herein as a “modifying moiety”
  • 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).
  • PTM amino acid side chain
  • PTM carboxyl or amino terminal end of the polypeptide chain
  • PTM alteration i.e., PTM alteration
  • 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.
  • 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.
  • a PTM can include both a covalent addition and non-covalent binding of a chemical moiety to a protein.
  • small ubiquitin-related modifiers SUMOs
  • SUMOs small ubiquitin-related modifiers
  • the covalent conjugation and non-covalent binding require different sequence motifs.
  • a PTM alteration can involve removal of a covalently conjugated or a non-covalently bound chemical moiety.
  • 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.
  • DAB deubiquitination
  • dephosphorylation deglycosylation
  • deacetylation deacetylation
  • deS-nitrosylation denitrosylation
  • 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:
  • phosphorylation refers to the addition of a phosphate (PO 4 ) group to a protein or other organic molecule.
  • dephosphorylation refers to the removal of a phosphate group from a protein or other organic molecule.
  • SUMO Small Ubiquitin-like Modifier
  • SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination.
  • deumoylation refers to the process whereby SUMO proteins are removed from proteins in cells.
  • 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.
  • ADP-ribosylation refers to the PTM of proteins that involves the addition of one or more ADP and ribose moieties.
  • de-ADP-ribosylation refers to the removal of one or more ADP and ribose moieties.
  • glycosylation refers to the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules.
  • 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.
  • deglycosylation refers to the enzymatic process that removes saccharides attached to proteins, lipids, or other organic molecules.
  • acetylation refers to the reaction that introduces an acetyl functional group into a chemical compound, and includes N-alpha-terminal acetylation and lysine acetylation.
  • deacetylation refers to the reaction that removes an acetyl functional group from a chemical compound.
  • S-nitrosylation or “nitrosylation” refer to the addition of a nitroso group to a sulfur atom of an amino acid residue of a protein.
  • 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.
  • “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.
  • “decitrullination” or “de-deimination” are the terms used for the removal of the amino acid citrulline from a protein.
  • 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.
  • 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 C ⁇ atom, forming hydroxyproline (Hyp).
  • proline may be hydroxylated instead on its C ⁇ atom.
  • Lysine may also be hydroxylated on its C ⁇ atom, forming hydroxylysine (Hyl).
  • dehydroxylation refers to the chemical process that removes one or more hydroxyl groups (—OH) from a protein.
  • ubiquitinlation refers to the process whereby the ubiquitin-like modifier Ufm-1 is covalently attached to a protein.
  • deufmylation refers to the process whereby the ubiquitin-like modifier Ufm-1 is removed from a protein.
  • fattenylation refers to the process whereby the ubiquitin-like modifier FAT10 is covalently attached to a protein.
  • defattenylation refers to the process whereby the ubiquitin-like modifier FAT10 is removed from a protein.
  • prenylation 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.
  • deprenylation refers to the removal of hydrophobic molecules from a protein.
  • 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.
  • S-palmitoylation refers to the covalent attachment of fatty acids, such as palmitic acid, to cysteine residues of proteins.
  • de-S-palmitoylation refers to the removal of fatty acids, such as palmitic acid, to cysteine residues from proteins.
  • tyrosine sulfation is a PTM where a sulfate group is added to a tyrosine residue of a protein molecule.
  • tyrosine desulfation is a PTM alteration where a sulfate group is removed from a tyrosine residue of a protein molecule.
  • “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.
  • formylation is a type of PTM in which a formyl group is added to the N-terminus of a protein.
  • deformylation is a type of PTM alteration in which a formyl group is removed from the N-terminus of a protein.
  • 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.
  • decarboxylation is a PTM alteration in which a carboxylic acid group is removed from glutamate residues in proteins.
  • 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).
  • 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.
  • a patient sample such as plasma, CSF, or from an extract prepared from biopsy tissue.
  • biomarkers of diseases such as Alzheimer's disease or cancer
  • Detecting PTMs of a large number of proteins provides a detailed fingerprint of the PTM enzymes released from tissues during disease.
  • the functional cell extract for use in the methods described herein is obtained from a biological sample.
  • 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.
  • CSF cerebrospinal fluid
  • 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.
  • 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.
  • 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.
  • a cell sample can be a heterogenous population of cells, such as the population of immune cells found in the spleen.
  • 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.
  • the functional cell extract can be directly prepared from a tissue or tumor by homogenization of the tissue or tumor.
  • the tumor sample refers to a biopsy of a tumor.
  • 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
  • intracellular proteins such as interstitial fluids, lymph, CSF, blood, serum, plasma, urine, saliva, umbilical cord blood, amniotic fluid, breast milk, mucus, semen, and vaginal secretions
  • intracellular proteins in such fluids are known.
  • 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.
  • PTM enzymes 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.
  • the invention now provides a means to assay PTM enzyme activities in samples that were previously not used for such analysis.
  • 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.
  • 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).
  • 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).
  • 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.
  • vitrification can be performed to prepare the cryopreserved biological sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an essentially detergent-free functional cell extract is contacted with a solid state array for detecting a PTM or PTM alteration.
  • a functional cell extract is prepared from a biological sample using one or more detergent-free or essentially detergent-free solutions.
  • 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 ⁇ l of a bodily fluid such as plasma or cerebrospinal fluid is sufficient for preparing an extract to cover a single 1 ⁇ 3 inch microarray.
  • cells are first harvested using standard techniques for collecting cells, e.g., from culture or from a specimen obtained from a patient.
  • 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.
  • 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 MgCl 2 , 5 mM KCl, 1 mM DTT, optionally containing a preferred mixture of protease inhibitors, such as COMPLETETM protease inhibitors (Roche).
  • a buffer such as 25 mM HEPES, pH 7.5, containing 1.5 mM MgCl 2 , 5 mM KCl, 1 mM DTT, optionally containing a preferred mixture of protease inhibitors, such as COMPLETETM protease inhibitors (Roche).
  • 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.
  • a ratio of about 0.5 to 1 volume of lysis solution to cell volume can be used to concentrate the functional cell extract.
  • 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.
  • the crude cell extract can be treated to remove membranes and whole or fragmented cells, such as by centrifugation.
  • 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.
  • 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.
  • 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.
  • the term “subject” refers to that specific animal.
  • 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.
  • subject also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish.
  • 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.
  • 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.
  • an extract can be supplemented with a reagent, a substrate, an enzyme, an enzyme inhibitor, a drug, an antibody, or any mixture thereof.
  • 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.
  • an additional cellular energy source in the form of, for example, ATP is provided to the functional cell extract.
  • 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
  • Creatine phosphokinase can also be added in addition to creatine phosphate, but may be omitted if sufficiently present in the extract or fluid.
  • a substrate for a PTM enzyme such as ubiquitin, is also added to the extract or fluid to establish a specific PTM reaction.
  • 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.
  • 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.
  • 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.
  • solid state array refers to any combination of one or more target proteins or peptides attached to a solid support.
  • 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.
  • 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.
  • 2-10 PTM or PTM alterations are identified simultaneously.
  • 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.
  • the multiple PTMs, PTM alterations, or combinations thereof are detected in parallel.
  • multiple PTMs, PTM alterations, or combinations thereof are detected sequentially.
  • the first PTM may affect the second PTM.
  • 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.
  • 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.
  • 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.
  • 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.
  • recombinant proteins bearing a tag such as a GST, FLAG, or myc tag
  • 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.
  • the recombinant proteins are directly deposited at specific locations in a microtiter plate, and binding is mediated by the properties of the microtiter plate.
  • untreated and irradiated polystyrene microtiter plates permit hydrophobic and hydrophilic interactions between the polystyrene and the protein being deposited.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • beads are dyed with fluorochromes having different fluorescence intensities.
  • the beads are 7.5 ⁇ m in diameter.
  • 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.
  • a target of an E1 ligase For example, a target of an E1 ligase.
  • a “capture bead” is a bead having a unique fluorescence emission intensity conjugated to a specific target protein.
  • 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.
  • 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).
  • a typical data acquisition and analysis package for e.g., BD CellQuestTM 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.
  • MFI median fluorescence intensities
  • 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.
  • 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.
  • 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.
  • a substrate e.g., ubiquitin
  • 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.
  • the microarray can include a large number of human proteins if the patient is a human patient.
  • 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.
  • the proteins in the microarray have various states of PTM; such proteins can be further modified by a functional extract, providing differential modification signals.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a modification moiety can be labeled with a labeling moiety such as biotin or a 35 S-labeled or radioiodinated amino acid.
  • Phosphorylation of proteins can be detected using an antibody specific for a phosphoprotein or by adding gamma- 32 P-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.
  • biochemical analysis e.g., by mass spectrometry
  • 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.
  • 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.).
  • 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
  • mass spectrometry refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m/z.”
  • 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.”
  • 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”).
  • m mass-to-charge ratio
  • z charge
  • 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.
  • Atmospheric Pressure Chemical Ionisation APCI
  • Chemical Ionisation CI
  • Electron Impact EI
  • Electrospray Ionisation ESI
  • FAB Field Desorption/Field Ionisation
  • MALDI Matrix Assisted Laser Desorption Ionisation
  • TSP Thermospray Ionisation
  • a gas phase ion spectrophotometer is used.
  • laser-desorption/ionization mass spectrometry is used to analyze the sample.
  • LLI-MS Modern laser desorption/ionization mass spectrometry
  • MALDI matrix assisted laser desorption/ionization
  • SELDI surface-enhanced laser desorption/ionization
  • MALDI matrix assisted laser desorption/ionization
  • 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).
  • the substrate surface is modified so that it is an active participant in the desorption process.
  • the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein modification of interest.
  • the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser.
  • 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.
  • 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.
  • a SELDI affinity surface to capture an analyte
  • adding matrix-containing liquid to the captured analyte to provide the energy absorbing material.
  • 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.
  • 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.
  • 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.
  • 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
  • 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%.
  • 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.
  • control spots e.g., spotted with buffer but no protein or of GST spotted on the array
  • 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).
  • 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.
  • 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 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.
  • 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.
  • 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
  • SUMOylation Cancer/tumor Ubc9 (E2 conjugating 35 A role for Ubc9 in enzyme) tumorigenesis Ubiquitination Alzheimers disease Bcl-2
  • Inhibition of the ubiquitin- proteasome system in Alzheimer's Disease Glycosylation Alzheimers disease tau
  • 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, ⁇ - 38 Parkin mediated lysine 63- ubiquitination synuclein
  • UCHL1 linked polyubiquitination a link to protein inclusions formation in Parkinson's and other conformational diseases?
  • 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.
  • 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.
  • 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.
  • SUMOylation Huntington disease Huntingtin (Httex1p) 52 SUMO modification of Huntingtin and Huntington's disease pathology.
  • SUMOylation Neuronal Intranuclear SUMOylation 54 SUMOylation substrates in Inclusion disease (NIID) substrates: neuronal intranuclear Promyelocytic inclusion disease.
  • NIID Inclusion disease
  • 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.
  • the ubiquitin proteasome Alzheimer's, L1, DJ-1 binds to the system in Huntington's, Prion and SUMO E3 PIASx, A ⁇ neurodegenerative amyotrophic lateral and tau, UBB + 1etc . . .
  • DJ-1 transcriptionally up- factor 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.
  • Phophorylation tumorigenesis 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.
  • Ubiquitination Fanconi anemia FANCD2 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 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.
  • HIV LAV/HTLV-III
  • 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.
  • 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.
  • MBP Systemic lupus basic protein
  • 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.
  • 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).
  • 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.
  • the functional extract can be supplemented with additional enzymes in different combinations in parallel assays.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the degradation of proteins involved in mitosis was examined by determining the polyubiquitination state of certain proteins at specific stages of the cell cycle.
  • 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.
  • APC Anaphase Promoting Complex
  • 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.
  • 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).
  • 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.
  • PBS phosphate buffered saline
  • 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
  • CP-extracts were then harvested, washed with PBS, lysed in Swelling Buffer (25 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM dithiothreitol, 1 tablet of Complete protease inhibitors (Roche)), and homogenized by freeze-thawing and passage through a needle.
  • Swelling Buffer 25 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM dithiothreitol, 1 tablet of Complete protease inhibitors (Roche)
  • Swelling Buffer 25 mM HEPES pH 7.5, 1.5 mM Mg
  • 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 ⁇ l) was supplemented with Degradation Cocktail (1 ⁇ L) 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 MgCl 2 ).
  • Degradation Cocktail (1 ⁇ L) 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 MgCl 2 ).
  • the arrays were then washed and incubated overnight with anti-polyubiquitin antibody (FK1, 1 mg/ml; Biomol) diluted 1:250.
  • anti-polyubiquitin antibody FK1, 1 mg/ml; Biomol
  • an anti-mouse Cy3-conjugated secondary antibody 3 ⁇ l; 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.
  • 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).
  • Coupled in vitro transcription and translation were performed from pCS2+ constructs using a rabbit reticulocyte lysate system (TnT SP6, Promega) or wheat germ extracts.
  • 35 S-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.
  • 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 35 S 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.
  • FIG. 5 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.
  • 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.
  • SNR Signal to Noise Ratio
  • 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.
  • 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.
  • 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.
  • samples are thawed on ice, supplemented with 5 ⁇ M ubiquitin, 2 mM ATP, and 150 mM creatine phosphate, and then incubated on a microarray to carry out the ubiquitination reaction.
  • E1 and/or E2 enzymes can be added to the extract, to determine if they are limiting the ubiquitination reaction.
  • 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.
  • PTM ubiquitination
  • 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.
  • terms associated with stress response (second row) showed no difference percentage of appearance in the ubiquitinated list when compared to the background list.
  • 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 ⁇ M) 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).
  • CSF ubiqutinate proteins
  • the CSF sample was supplemented with 2 mM ATP and 150 mM creatine phosphate and ubiquitin (5 ⁇ M).
  • the sample was then incubated on a Human PROTO-ARRAY® (Invitrogen) protein microarray in order to identify the basal ubiquitination activity in the sample.
  • 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 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, +).
  • 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 ⁇ M 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 ⁇ m). 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.
  • 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.
  • 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_15
  • 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
  • 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 CREAT
  • 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_G
  • 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 ARRDC
  • 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 BC
  • 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 CH
  • 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 PROTE
  • 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 (
  • 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, MEM
  • 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
  • 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.
  • the highest reactivities was UbcH9, the only known E2 characterized to date for sumo conjugation.
  • 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.
  • the top reacting proteins for each of these modifications were the enzymes that are involved in catalysis of the relevant PTM itself.
  • modifying moieties 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.
  • FDR false discovery rate
  • modifying moiety tested i.e. ubiquitin, sumol, sumo2/3, nedd8, FA10, UFM1, ISG15
  • FIG. 15 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.

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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.
  • 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.
  • 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
    SNEVPrp19/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.
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  • 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 (23)

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.
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