Reagents for the detection of protein phosphorylation in carcinoma signaling pathways

Information

  • Patent Application
  • 20100151495
  • Publication Number
    20100151495
  • Date Filed
    February 29, 2008
    16 years ago
  • Date Published
    June 17, 2010
    14 years ago
Abstract
The invention discloses nearly 443 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human carcinoma, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Protein kinases (including Serine/Threonine dual specificity, and Tyrosine kinases), Adaptor/Scaffold proteins, Transcription factors, Phospoatases, Tumor supressors, Ubiquitin conjugating system proteins, Translation initiation complex proteins, RNA binding proteins, Apoptosis proteins, Adhesion proteins, G protein regulators/GTPase activating protein/Guanine nucleotide exchange factor proteins, and DNA binding/replication/repair proteins, as well as other protein types.
Description
FIELD OF THE INVENTION

The invention relates generally to antibodies and peptide reagents for the detection of protein phosphorylation, and to protein phosphorylation in cancer.


BACKGROUND OF THE INVENTION

The activation of proteins by post-translational modification is an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. Protein phosphorylation, for example, plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes. Yet, in spite of the importance of protein modification, it is not yet well understood at the molecular level, due to the extraordinary complexity of signaling pathways, and the slow development of technology necessary to unravel it.


Protein phosphorylation on a proteome-wide scale is extremely complex as a result of three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome, for example, encodes over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Most kinases phosphorylate many different substrate proteins, at distinct tyrosine, serine, and/or threonine residues. Indeed, it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases.


Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra. Understanding which proteins are modified by these kinases will greatly expand our understanding of the molecular mechanisms underlying oncogenic transformation. Therefore, the identification of, and ability to detect, phosphorylation sites on a wide variety of cellular proteins is crucially important to understanding the key signaling proteins and pathways implicated in the progression of diseases like cancer.


Carcinoma is one of the two main categories of cancer, and is generally characterized by the formation of malignant tumors or cells of epithelial tissue original, such as skin, digestive tract, glands, etc. Carcinomas are malignant by definition, and tend to metastasize to other areas of the body. The most common forms of carcinoma are skin cancer, lung cancer, breast cancer, and colon cancer, as well as other numerous but less prevalent carcinomas. Current estimates show that, collectively, various carcinomas will account for approximately 1.65 million cancer diagnoses in the United States alone, and more than 300,000 people will die from some type of carcinoma during 2005. (Source: American Cancer Society (2005)). The worldwide incidence of carcinoma is much higher.


As with many cancers, deregulation of receptor tyrosine kinases (RTKs) appears to be a central theme in the etiology of carcinomas. Constitutively active RTKs can contribute not only to unrestricted cell proliferation, but also to other important features of malignant tumors, such as evading apoptosis, the ability to promote blood vessel growth, the ability to invade other tissues and build metastases at distant sites (see Blume-Jensen et al., Nature 411: 355-365 (2001)). These effects are mediated not only through aberrant activity of RTKs themselves, but, in turn, by aberrant activity of their downstream signaling molecules and substrates.


The importance of RTKs in carcinoma progression has led to a very active search for pharmacological compounds that can inhibit RTK activity in tumor cells, and more recently to significant efforts aimed at identifying genetic mutations in RTKs that may occur in, and affect progression of, different types of carcinomas (see, e.g., Bardell et al., Science 300: 949 (2003); Lynch et al., N. Eng. J. Med. 350: 2129-2139 (2004)). For example, non-small cell lung carcinoma patients carrying activating mutations in the epidermal growth factor receptor (EGFR), an RTK, appear to respond better to specific EGFR inhibitors than do patients without such mutations (Lynch et al., supra.; Paez et al., Science 304:1497-1500 (2004)).


Clearly, identifying activated RTKs and downstream signaling molecules driving the oncogenic phenotype of carcinomas would be highly beneficial for understanding the underlying mechanisms of this prevalent form of cancer, identifying novel drug targets for the treatment of such disease, and for assessing appropriate patient treatment with selective kinase inhibitors of relevant targets when and if they become available.


However, although a few key RTKs involved in carcinoma progression are known, there is relatively scarce information about kinase-driven signaling pathways and phosphorylation sites that underly the different types of carcinoma. Therefore there is presently an incomplete and inaccurate understanding of how protein activation within signaling pathways is driving these complex cancers. Accordingly, there is a continuing and pressing need to unravel the molecular mechanisms of kinase-driven oncogenesis in carcinoma by identifying the downstream signaling proteins mediating cellular transformation in these cancers. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho-specific antibodies and AQUA peptides, to detect and quantify them remains especially important to advancing our understanding of the biology of this disease.


Presently, diagnosis of carcinoma is made by tissue biopsy and detection of different cell surface markers. However, misdiagnosis can occur since some carcinoma cases can be negative for certain markers and because these markers may not indicate which genes or protein kinases may be deregulated. Although the genetic translocations and/or mutations characteristic of a particular form of carcinoma can be sometimes detected, it is clear that other downstream effectors of constitutively active kinases having potential diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phosphorylation sites involved in different types of carcinoma and development of new reagents to detect and quantify these sites and proteins may lead to improved diagnostic/prognostic markers, as well as novel drug targets, for the detection and treatment of this disease.


SUMMARY OF THE INVENTION

The invention discloses nearly 443 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human carcinomas and provides new reagents, including phosphorylation-site specific antibodies and AQUA peptides, for the selective detection and quantification of these phosphorylated sites/proteins. Also provided are methods of using the reagents of the invention for the detection, quantification, and profiling of the disclosed phosphorylation sites.





BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Is a diagram broadly depicting the immunoaffinity isolation and mass-spectrometric characterization methodology (IAP) employed to identify the novel phosphorylation sites disclosed herein.


FIG. 2—Is a table (corresponding to Table 1) enumerating the 443 carcinoma signaling protein phosphorylation sites disclosed herein: Column A=the name of the parent protein; Column B=the SwissProt accession number for the protein (human sequence); Column C=the protein type/classification; Column D=the tyrosine residue (in the parent protein amino acid sequence) at which phosphorylation occurs within the phosphorylation site; Column E=the phosphorylation site sequence encompassing the phosphorylatable residue (residue at which phosphorylation occurs (and corresponding to the respective entry in Column D) appears in lowercase; Column F=the type of carcinoma in which the phosphorylation site was discovered; Column G=the cell type(s) in which the phosphorylation site was discovered; and Column H=the SEQ ID NO.


FIG. 3—is an exemplary mass spectrograph depicting the detection of the tyrosine 1048 phosphorylation site in flt 1 (see Row 164 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2).


FIG. 4—is an exemplary mass spectrograph depicting the detection of the tyrosine 2556 phosphorylation site in NF1 (see Row 128 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2).


FIG. 5—is an exemplary mass spectrograph depicting the detection of the tyrosine 315 phosphorylation site in OCLN (see Row 44 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2) and M# (and lowercase “m”) indicates an oxidized methionine also detected.


FIG. 6—is an exemplary mass spectrograph depicting the detection of the tyrosine 1200 phosphorylation site in PHLPP (see Row 193 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2).


FIG. 7—is an exemplary mass spectrograph depicting the detection of the tyrosine 366 phosphorylation site in TNS1 (see Row 20 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2).


FIG. 8—is an exemplary mass spectrograph depicting the detection of the tyrosine 188 phosphorylation site in Yap1 (see Row 328 in FIG. 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (shown as lowercase “y” in FIG. 2).





DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, nearly 443 novel protein phosphorylation sites in signaling proteins and pathways underlying carcinoma have now been discovered. These newly described phosphorylation sites were identified by employing the techniques described in “Immunoaffinity Isolation of Modified Peptides From Complex Mixtures,” U.S. Patent Publication No. 20030044848, Rush et al., using cellular extracts from a variety of human carcinoma-derived cell lines, such as 3T3-abl, U118 MG, 293T, NCI-N87, A549, etc., as further described below. The novel phosphorylation sites (tyrosine), and their corresponding parent proteins, disclosed herein are listed in Table 1.


These phosphorylation sites correspond to numerous different parent proteins (the full sequences of which (human) are all publicly available in SwissProt database and their Accession numbers listed in Column B of Table 1/FIG. 2), each of which fall into discrete protein type groups, for example Protein Kinases (Serine/Threonine nonreceptor, Tyrosine receptor, Tyrosine nonreceptor, dual specificity and other), Adaptor/Scaffold proteins, transcription factors, phosphates, tumor suppressors, etc. (see Column C of Table 1), the phosphorylation of which is relevant to signal transduction activity underlying carcinomas (e.g., skin, lung, breast and colon cancer), as disclosed herein.


The discovery of the nearly 443 novel protein phosphorylation sites described herein enables the production, by standard methods, of new reagents, such as phosphorylation site-specific antibodies and AQUA peptides (heavy-isotope labeled peptides), capable of specifically detecting and/or quantifying these phosphorylated sites/proteins. Such reagents are highly useful, inter alia, for studying signal transduction events underlying the progression of carcinoma. Accordingly, the invention provides novel reagents—phospho-specific antibodies and AQUA peptides—for the specific detection and/or quantification of a Carcinoma-related signaling protein/polypeptide only when phosphorylated (or only when not phosphorylated) at a particular phosphorylation site disclosed herein. The invention also provides methods of detecting and/or quantifying one or more phosphorylated Carcinoma-related signaling proteins using the phosphorylation-site specific antibodies and AQUA peptides of the invention, and methods of obtaining a phosphorylation profile of such proteins (e.g. Kinases).


In part, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a given Carcinoma-related signaling protein only when phosphorylated (or not phosphorylated, respectively) at a particular tyrosine enumerated in Column D of Table 1/FIG. 2 comprised within the phosphorylatable peptide site sequence enumerated in corresponding Column E. In further part, the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the detection and quantification of a given Carcinoma-related signaling protein, the labeled peptide comprising a particular phosphorylatable peptide site/sequence enumerated in Column E of Table 1/FIG. 2 herein. For example, among the reagents provided by the invention is an isolated phosphorylation site-specific antibody that specifically binds the KIAA2002 kinase (serine/threonine) only when phosphorylated (or only when not phosphorylated) at tyrosine 635 (see Row 155 (and Columns D and E) of Table 1/FIG. 2). By way of further example, among the group of reagents provided by the invention is an AQUA peptide for the quantification of phosphorylated KIAA2002 kinase, the AQUA peptide comprising the phosphorylatable peptide sequence listed in Column E, Row 155 of Table 1/FIG. 2 (which encompasses the phosphorylatable tyrosine at position 635).


In one embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a human Carcinoma-related signaling protein selected from Column A of Table 1 (Rows 2-444) only when phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1, 3-8, 10-20, 22-24, 26-63, 65-67, 69-92, 94-154, 156-225, 227-243, 245-302, 304-325, 327-332, 334-340, 342-360, 362-365, 368-408, 411-432, and 434-443), wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine. In another embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a Carcinoma-related signaling protein selected from Column A of Table 1 only when not phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1, comprised within the peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1, 3-8, 10-20, 22-24, 26-63, 65-67, 69-92, 94-154, 156-225, 227-243, 245-302, 304-325, 327-332, 334-340, 342-360, 362-365, 368-408, 411-432, and 434-443), wherein said antibody does not bind said signaling protein when phosphorylated at said tyrosine. Such reagents enable the specific detection of phosphorylation (or non-phosphorylation) of a novel phosphorylatable site disclosed herein. The invention further provides immortalized cell lines producing such antibodies. In one preferred embodiment, the immortalized cell line is a rabbit or mouse hybridoma.


In another embodiment, the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein selected from Column A of Table 1, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1, 3-8, 10-20, 22-24, 26-63, 65-67, 69-92, 94-154, 156-225, 227-243, 245-302, 304-325, 327-332, 334-340, 342-360, 362-365, 368-408, 411-432, and 434-443), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D of Table 1. In certain preferred embodiments, the phosphorylatable tyrosine within the labeled peptide is phosphorylated, while in other preferred embodiments, the phosphorylatable residue within the labeled peptide is not phosphorylated.


Reagents (antibodies and AQUA peptides) provided by the invention may conveniently be grouped by the type of Carcinoma-related signaling protein in which a given phosphorylation site (for which reagents are provided) occurs. The protein types for each respective protein (in which a phosphorylation site has been discovered) are provided in Column C of Table 1/FIG. 2, and include: Actin binding proteins, Adaptor/Scaffold proteins, Adhesion proteins, Apoptosis proteins, Cell Cycle Regulation proteins, Cell surface proteins, Channel proteins, Chaperone proteins, Cytoskeleton proteins, DNA binding proteins, DNA repair proteins, DNA replication proteins, Enzymes, Extracellular Matrix proteins, G protein regulatory proteins, GTPase activating proteins, Guanine nucleotide exchange factor proteins, Helicase proteins, Hydrolase proteins, Inhibitor proteins, Kinases (Serine/Threonine, dual specificity, Tyrosine etc.), Lipid binding proteins, Mitochondrial proteins, Motor proteins, Myosin biding proteins, Phosphatase proteins, Oxidoreductase proteins, Phospholipases, Proteases, Receptor proteins, RNA binding proteins, Secreted proteins, Transcription factor proteins, Transcription initiator complex proteins, Transcription coactivator/corepressor proteins, Transferase proteins, Translation initiation complex proteins, Transporter proteins, Tumor suppressor proteins, Ubiquitin conjugating proteins, and Vesicle proteins. Each of these distinct protein groups is considered a preferred subset of Carcinoma-related signal transduction protein phosphorylation sites disclosed herein, and reagents for their detection/quantification may be considered a preferred subset of reagents provided by the invention.


Particularly preferred subsets of the phosphorylation sites (and their corresponding proteins) disclosed herein are those occurring on the following protein types/groups listed in Column C of Table 1/FIG. 2: 1) Protein kinases (including Serine/Threonine dual specificity, and Tyrosine kinases), 2) Adaptor/Scaffold proteins, 3) Transcription factors, 4) Phospoatases, 5) Tumor supressors, 6) Ubiquitin conjugating system proteins, 7) Translation initiation complex proteins, 8) RNA binding proteins, 9) Apoptosis proteins, 10) Adhesion proteins, 11) G protein regulators/GTPase activating protein/Guanine nucleotide exchange factor proteins, and 12) DNA binding/replication/repair proteins. Accordingly, among preferred subsets of reagents provided by the invention are isolated antibodies and AQUA peptides useful for the detection and/or quantification of the foregoing preferred protein/phosphorylation site subsets.


In one subset of preferred embodiments there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Protein kinase selected from Column A, Rows 138-165, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 138-165, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 138-165, of Table 1 (SEQ ID NOs: 137-154, and 156-164), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Protein kinase when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Protein kinase selected from Column A, Rows 138-165, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 138-165, of Table 1 (SEQ ID NOs: 137-154, and 156-164), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 138-165, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Protein kinase phosphorylation sites are particularly preferred: PIK3CB (Y436), ILK (Y351), IRAK1 (Y395), KIAA2002 (Y635), and FLT1 (Y1048), (see SEQ ID NOs: 138, 145, 146, 154, and 163).


In one subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds an Adaptor/Scaffold protein selected from Column A, Rows 5-26, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 5-26, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 5-26, of Table 1 (SEQ ID NOs: 4-8, 10-20, and 22-24), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Adaptor/Scaffold protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is an Adaptor/Scaffold protein selected from Column A, Rows 5-26, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 5-26, of Table 1 (SEQ ID NOs: 4-8, 10-20, and 22-24), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 5-26, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Adaptor/Scaffold protein phosphorylation site is particularly preferred: TNS1 (Y366), (see SEQ ID NO: 19).


In another subset of preferred embodiments there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Transcription factor protein selected from Column A, Rows 266-330, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 266-330, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 266-330, of Table 1 (SEQ ID NOs: 265-302, 304-325, and 327-329), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Transcription factor protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Transcription factor protein selected from Column A, Rows 266-330, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 266-330, of Table 1 (SEQ ID NOs: 265-302, 304-325, and 327-329), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 266-330, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Transcription factor protein phosphorylation sites are particularly preferred: HIC1 (Y136), MLL (Y2136), TBX1 (Y38), TBX5 (Y114), and YAP1 (Y188) (see SEQ ID NOs: 271, 276, 289, 291, and 327).


In still another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Phosphatases selected from Column A, Rows 192-200, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 192-200, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 192-200, of Table 1 (SEQ ID NOs: 191-199), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Phosphatase proteins when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Phosphatase selected from Column A, Rows 192-200, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 192-200, of Table 1 (SEQ ID NOs: 191-199), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 192-200, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Phosphatase phosphorylation sites are particularly preferred: PHLPP (Y1200), PTPN11 (Y263) and PTPRT (Y1003) (see SEQ ID NOs: 192, 194 and 197).


In still another subset of preferred embodiments there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Tumor suppressor protein selected from Column A, Rows 396-402, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 396-402, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 396-402, of Table 1 (SEQ ID NOs: 395-401), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Tumor suppressor protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Tumor suppressor protein selected from Column A, Rows 396-402, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 396-402, of Table 1 (SEQ ID NOs: 395-401), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 396-402, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Tumor suppressor phosphorylation sites are particularly preferred: APC (Y737), RB1 (Y239), and TP53 (Y327) (see SEQ ID NOs: 395, 398 and 401).


In still another subset of preferred embodiments there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Ubiquitin conjugating system protein selected from Column A, Rows 403-422, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 403-422, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 403-422, of Table 1 (SEQ ID NOs: 402-408, and 411-421), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Ubiquitin conjugating system protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Ubiquitin conjugating system protein selected from Column A, Rows 403-422, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 403-422, of Table 1 (SEQ ID NOs: 402-408, and 411-421), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 403-422, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Ubiquitin conjugating system protein phosphorylation sites are particularly preferred: CUL2 (Y43), CUL5 (Y214), and NEDD4 (Y43) (see SEQ ID NOs: 404, 405, and 411).


In still another subset of preferred embodiments there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a Translation initiation complex protein selected from Column A, Rows 351-370, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 351-370, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-370 of Table 1 (SEQ ID NOs: 350-360, 362-365, and 368-369), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Translation initiation complex protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a Translation initiation complex protein selected from Column A, Rows 351-370, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-370, of Table 1 (SEQ ID NOs: 350-360, 362-365, and 368-369), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 351-370, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Translation initiation complex protein phosphorylation site is particularly preferred: EIF4B (Y105) (see SEQ ID NO: 358).


In still another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds an RNA binding protein selected from Column A, Rows 240-257, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 240-257, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 240-257, of Table 1 (SEQ ID NOs: 239-243, and 245-256), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the RNA binding protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is an RNA binding protein selected from Column A, Rows 240-257, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 240-257, of Table 1 (SEQ ID NOs: 239-243, and 245-256), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 240-257, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following RNA binding protein phosphorylation sites are particularly preferred: RAE1 (Y274) (see SEQ ID NO: 250).


In yet another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds an Apoptosis protein selected from Column A, Rows 58-60, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 58-60, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 58-60, of Table 1 (SEQ ID NOs: 57-59), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Apoptosis protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is an Apoptosis protein selected from Column A, Rows 58-60, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 58-60, of Table 1 (SEQ ID NOs: 57-59), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 58-60, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Apoptosis protein phosphorylation sites are particularly preferred: IFIH1 (Y1000) (see SEQ ID NO: 57).


In yet another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody specifically binds an Adhesion protein selected from Column A, Rows 27-57, of Table 1 only when phosphorylated at the tyrosine listed in corresponding to Column D, Rows 27-57, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 27-57, of Table 1 (SEQ ID NOs: 26-56), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Adhesion protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is an Adhesion protein selected from Column A, Rows 27-57, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 27-57, of Table 1 (SEQ ID NOs: 26-56), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 27-57, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following Adhesion protein phosphorylation sites are particularly preferred: F11R (Y280), OCLN (Y315) (see SEQ ID NOs: 33 and 43).


In yet another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a G protein regulator proteins/GTPase activating proteins/Guanine nucleotide exchange factor proteins selected from Column A, Rows 122-130, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 122-130, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 122-130, of Table 1 (SEQ ID NOs: 121-129), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the G protein regulator proteins/GTPase activating proteins/Guanine nucleotide exchange factor proteins when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a G protein regulator proteins/GTPase activating proteins/Guanine nucleotide exchange factor proteins selected from Column A, Rows 122-130, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 122-130, of Table 1 (SEQ ID NOs: 121-129), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 122-130, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following G protein regulator proteins/GTPase activating proteins/Guanine nucleotide exchange factor proteins phosphorylation sites are particularly preferred: NF1 (Y2556), RASGRP3 (Y523) (see SEQ ID NOs: 127 and 129).


In still another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds a DNA binding/replication/repair protein selected from Column A, Rows 95-104, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 95-104, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 95-104, of Table 1 (SEQ ID NOs: 94-103), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the DNA binding/replication/repair protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is a DNA binding/replication/repair protein selected from Column A, Rows 95-104, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 95-104, of Table 1 (SEQ ID NOs: 94-103), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 95-104, of Table 1.


Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following DNA binding/replication/repair protein phosphorylation sites are particularly preferred: SMARCA5 (Y719) (see SEQ ID NO: 95).


In still another subset of preferred embodiments, there is provided:


(i) An isolated phosphorylation site-specific antibody that specifically binds the Receptor protein of Row 218, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Row 218 of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Row 218 of Table 1 (SEQ ID NO: 217), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.


(ii) An equivalent antibody to (i) above that only binds the Receptor protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).


(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Carcinoma-related signaling protein that is the Receptor protein of Column A, Row 218, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Row 218 of Table 1 (SEQ ID NO: 217), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 217 of Table 1.


The invention also provides, in part, an immortalized cell line producing an antibody of the invention, for example, a cell line producing an antibody within any of the foregoing preferred subsets of antibodies. In one preferred embodiment, the immortalized cell line is a rabbit hybridoma or a mouse hybridoma.


In certain other preferred embodiments, a heavy-isotope labeled peptide (AQUA peptide) of the invention (for example, an AQUA peptide within any of the foregoing preferred subsets of AQUA peptides) comprises a disclosed site sequence wherein the phosphorylatable tyrosine is phosphorylated. In certain other preferred embodiments, a heavy-isotope labeled peptide of the invention comprises a disclosed site sequence wherein the phosphorylatable tyrosine is not phosphorylated.


The foregoing subsets of preferred reagents of the invention should not be construed as limiting the scope of the invention, which, as noted above, includes reagents for the detection and/or quantification of disclosed phosphorylation sites on any of the other protein type/group subsets (each a preferred subset) listed in Column C of Table 1/FIG. 2.


Also provided by the invention are methods for detecting or quantifying a Carcinoma-related signaling protein that is tyrosine phosphorylated, said method comprising the step of utilizing one or more of the above-described reagents of the invention to detect or quantify one or more Carcinoma-related signaling protein(s) selected from Column A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1. In certain preferred embodiments of the methods of the invention, the reagents comprise a subset of preferred reagents as described above.


Also provided by the invention is a method for obtaining a phosphorylation profile of protein kinases that are phosphorylated in Carcinoma signaling pathways, said method comprising the step of utilizing one or more isolated antibody that specifically binds a protein kinase selected from Column A, Rows 138-165, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 138-165, of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 138-165, of Table 1 (SEQ ID NOs: 137-154, and 156-164), to detect the phosphorylation of one or more of said protein kinases, thereby obtaining a phosphorylation profile for said kinases.


The identification of the disclosed nearly 443 novel Carcinoma-related signaling protein phosphorylation sites, and the standard production and use of the reagents provided by the invention are described in further detail below and in the Examples that follow.


All cited references are hereby incorporated herein, in their entirety, by reference. The Examples are provided to further illustrate the invention, and do not in any way limit its scope, except as provided in the claims appended hereto.










TABLE 1







Newly Discovered Carcinoma-Related



Signaling Protein Phosphorylation Sites.















A
B
C
D
E
H




Protein
Accession
Protein
Phospho
Phosphorylation


1
Name
No.
Type
Residue
Site Sequence
SEQ ID NO

















2
FSCN2
NP_036550.1
Actin binding protein
Y228
yLAPVGPAGTLKAGRNTR
SEQ ID NO: 1






3
TENC1

Actin binding protein
Y493
GPLDGSPyAQVQR
SEQ ID NO: 2





4
TENC1
NP_056134.2
Actin binding protein
Y780
AGEEGHEGCSyTMCPEGR
SEQ ID NO: 3





5
DLG5
NP_004738.3
Adaptor/scaffold
Y71
LAFATHGTAFDKRPyHR
SEQ ID NO: 4





6
DLG5
NP_004738.3
Adaptor/scaffold
Y1133
LSLDLSHRTCSDySEMR
SEQ ID NO: 5





7
IRS4
NP_003595.1
Adaptor/scaffold
Y743
GyMMMFPR
SEQ ID NO: 6





8
IRS4
NP_003595.1
Adaptor/scaffold
Y808
SWSSyFSLPNPFR
SEQ ID NO: 7





9
IRS4
NP_003595.1
Adaptor/scaffold
Y828
SSPLGQNDNSEyVPMLPGK
SEQ ID NO: 8





10
IRS4

Adaptor/scaffold
Y921
EADSSSDyVNMDFTK
SEQ ID NO: 9





11
KPNA5
NP_002260.2
Adaptor/scaffold
Y17
MDAMASPGKDNYRMKSyK
SEQ ID NO: 10





12
PARD3
NP_062565.2
Adaptor/scaffold
Y489
DVTIGGSAPIyVK
SEQ ID NO: 11





13
PARD3
NP_062565.2
Adaptor/scaffold
Y1310
KEQQMKKQPPSEGPSNyDSYK
SEQ ID NO: 12





14
RAPH1
NP_998754.1
Adaptor/scaffold
Y1226
AGYGGSHISGyATLR
SEQ ID NO: 13





15
SHANK2
NP_036441.1
Adaptor/scaffold
Y322
VyGTIKPAFNQNSAAK
SEQ ID NO: 14





16
SHANK2
NP_036441.1
Adaptor/scaffold
Y372
ELDRYSLDSEDLySR
SEQ ID NO: 15





17
SHANK2
NP_036441.1
Adaptor/scaffold
Y606
AQGPESSPAVPSASSGTAGPGNyVHPLT
SEQ ID NO: 16







GR





18
SORBS1
NP_006425.2
Adaptor/scaffold
Y555
GERITLLRQVDENWyEGR
SEQ ID NO: 17





19
TJP2
NP_004808.2
Adaptor/scaffold
Y426
HQYSDyDYHSSSEK
SEQ ID NO: 18





20
TNS1
NP_072174.3
Adaptor/scaffold
Y366
DDGMEEVVGHTQGPLDGSLyAK
SEQ ID NO: 19





21
TNS1
NP_072174.3
Adaptor/scaffold
Y1254
HPAGVyQVSGLHNK
SEQ ID NO: 20





22
TNS1

Adaptor/scaffold
Y1326
HVAYGGySTPEDR
SEQ ID NO 21





23
TRPC4AP
NP_056453.1
Adaptor/scaffold
Y603
FNKyINTDAKFQVFLKQINSSLVDSNML
SEQ ID NO: 22







VR





24
LPP
NP_005569.1
Adaptor/scaffold;
Y273
GGMDyAYIPPPGLQPEPGYGYAPNQGR
SEQ ID NO: 23





Cytoskeletal protein





25
FNBP1L
NP_060207.2
Adaptor/scaffold;
Y448
ESPEGSyTDDANQEVR
SEQ ID NO: 24





Unknown function





26
EPS15L1

Adaptor/scaffold;
Y564
SLEQyDQVLDGAHGASLTDLANLSEGVS
SEQ ID NO. 25





Vesicle protein

LAER





27
CDH3
NP_001784.2
Adhesion
Y713
DNVFYYGEEGGGEEOQDyDITQLHR
SEQ ID NO: 26





28
CDH3
NP_001784.2
Adhesion
Y823
KLADMyGGGEDD
SEQ ID NO: 27





29
CDH6
NP_004923.1
Adhesion
Y4
TyRYFLLLFWVGQPYPTLSTPLSK
SEQ ID NO: 28





30
CDH6
NP_004923.1
Adhesion
Y6
TYRyFLLLFWVGQPYPTLSTPLSK
SEQ ID NO: 29





31
DCBLD2
NP_563615.3
Adhesion
Y565
KTEGTyDLPYWDR
SEQ ID NO: 30





32
DSC3
NP_001932.1
Adhesion
Y493
IKENLAVGSKINGyK
SEQ ID NO: 31





33
ERBB2IP
NP_00100660
Adhesion
Y1021
SESTENQSyAKHSANMNFSNHNNVR
SEQ ID NO: 32




0.1





34
F11R
NP_058642.1
Adhesion
Y280
KVIySQPSAR
SEQ ID NO: 33





35
HSPG2
CAA44373.1
Adhesion
Y1711
GPHYFyWSREDGRPVPSGTQQR
SEQ ID NO: 34





36
ITGA2
NP_002194.1
Adhesion
Y1005
NPLMyLTGVQTDKAGDISCNADINPLKIG
SEQ ID NO: 35







QTSSSVSFK





37
ITGAM
NP_000623.2
Adhesion
Y283
EGVIRyVIGVGDAFRSEK
SEQ ID NO: 36





38
ITGBS
NP_002204.2
Adhesion
Y774
ARYEMASNPLyR
SEQ ID NO: 37





39
L1CAM
NP_076493.1
Adhesion
Y1151
ySVKDKEDTQVDSEARPMKDETFGEYS
SEQ ID NO: 38







DNEEK





40
LAMA4
NP_002281.1
Adhesion
Y1317
yELIVDKSR
SEQ ID NO: 39





41
MCAM
NP_006491.2
Adhesion
Y641
APGDQGEKyIDLRH
SEQ ID NO: 40





42
NRXN2
NP_055895.1
Adhesion
Y41
yARWAGAASSGELSFSLRTNATR
SEQ ID NO: 41





43
OCLN
NP_002529.1
Adhesion
Y287
SNILWDKEHIyDEQPPNVEEWVK
SEQ ID NO: 42





44
OCLN
NP_002529.1
Adhesion
Y315
NVSAGTQDVPSPPSDyVERVDSPMAYS
SEQ ID NO: 43







SNGK





45
OCLN
NP_002529.1
Adhesion
Y402
TEQDHYETDyTTGGESCDELEEDWIR
SEQ ID NO: 44





46
OCLN
NP_002529.1
Adhesion
Y443
NFDTGLQEyK
SEQ ID NO: 45





47
PCDH1
NP115796.2
Adhesion
Y1058
LQDPSQHSyYDSGLEE
SEQ ID NO: 46





48
PCDH20
NP_073754.1
Adhesion
Y883
VESVSCMPTLVALSVISLGSITLVTGMGIy
SEQ ID NO: 47







ICLRK





49
PCDHB15
NP_061758.1
Adhesion
Y279
DLDTGTNGEISySLYYSSQEIDK
SEQ ID NO: 48





50
PCDHB15
NP_061758.1
Adhesion
Y282
DLDTGTNGEISYSLyYSSQEIDK
SEQ ID NO: 49





51
PCDHB15
NP_061758.1
Adhesion
Y283
DLDTGTNGEISYSLYySSQEIDK
SEQ ID NO: 50





52
PKP3
NP_009114.1
Adhesion
Y390
NLIyDNADNK
SEQ ID NO: 51





53
PVRL4
NP_112178.1
Adhesion
Y502
KPTGNGIyINGR
SEQ ID NO: 52





54
DSG2
NP_001934.1
Adhesion; Calcium-
Y967
VyAPASTLVDQPYANEGTVVVTER
SEQ ID NO: 53





binding protein





55
DSG2
NP_001934.1
Adhesion; Calcium-
Y978
VYAPASTLVDQPyANEGTVVVTER
SEQ ID NO: 54





binding protein





56
DSG2
NP_001934.1
Adhesion; Calcium-
Y1060
VLAPASTLQSSyQIPTENSMTAR
SEQ ID NO: 55





binding protein





57
PTPNS1
NP542970.1
Adhesion; Cell surface;
Y429
EITQDTNDITyADLNLPK
SEQ ID NO: 56





Receptor, misc.





58
IFIH1
NP_071451.2
Apoptosis
Y1000
KQyKKWVELPITFPNLDYSECCLFSDED
SEQ ID NO: 57





59
IFIH1
NP_071451.2
Apoptosis
Y1015
KQYKKWVELPITFPNLDySECCLFSDED
SEQ ID NO: 58





60
MAEA
NP_00101740
Apoptosis
Y19
MTLKVQEyPTLKVPYETLNKR
SEQ ID NO: 59




5.1





61
LLGL2
NP_004515.2
Cell cycle regulation
Y499
VGSFDPySDDPR
SEQ ID NO: 60





62
MSH4
NP_002431.2
Cell cycle regulation
Y889
AVyHLATRLVQTAR
SEQ ID NO: 61





63
SYCP2
NP_055073.2
Cell cycle regulation
Y1453
EFVDFWEKIFQKFSAyQK
SEQ ID NO: 62





64
TACC2
NP_008928.1
Cell cycle regulation
Y804
EAAHPTDVSISKTALySR
SEQ ID NO: 63





65
CSPG6

Cell cycle regulation;
Y669
GALTGGYyDTR
SEQ ID NO: 64





DNA repair





66
HEM1
NP_056416.2
Cell surface
Y315
VTEDLFSSLKGyGKRVADIK
SEQ ID NO: 65





67
KM-HN-1
NP689988.1
Cell surface
Y790
ICNQHNDPSKTTyISR
SEQ ID NO: 66





68
M11S1
NP_005889.3
Cell surface
Y449
GYTASQPLyQPSHATE
SEQ ID NO: 67





69
MUC13

Cell surface
Y500
DSQMQNPySR
SEQ ID NO: 68





70
MUC13
NP_149038.2
Cell surface
Y511
HSSMPRPDy
SEQ ID NO:69





71
ROM1
NP_000318.1
Cell surface
Y288
yLQTALEGLGGVIDAGGETQGYLFPSG
SEQ ID NO: 70







LK





72
ROM1
NP_000318.1
Cell surface
Y309
LQTALEGLGGVIDAGGETQGyLFPSG
SEQ ID NO: 71







LK





73
SLITRK6
NP_115605.2
Cell surface
Y805
LMETLMySRPR
SEQ ID NO: 72





74
SLITRK6
NP_115605.2
Cell surface
Y820
KVLVEQTKNEyFELK
SEQ ID NO: 73





75
RYR3
NP_001027.2
Channel, calcium
Y2824
LEDDPLyTSYSSMMAK
SEQ ID NO: 74





76
CLCN1
NP_000074.1
Channel, chloride
Y686
LRAAQEMARKLSELPyDGKAR
SEQ ID NO: 75





77
GJA1
NP_000156.1
Channel, misc.
Y313
QASEQNWANySAEQNR
SEQ ID NO: 76





78
KCNQ3
NP_004510.1
Channel, potassium
Y502
GyGNDFPIEDMIPTLK
SEQ ID NO: 77





79
TBCE
NP_003184.1
Chaperone
Y493
LLKVPVSDLLLSyESPKK
SEQ ID NO: 78





80
EPB41L1
NP_036288.2
Cytoskeletal protein
Y864
AVVyRETDPSPEER
SEQ ID NO 79





81
EPB41L4A
NP_071423.3
Cytoskeletal protein
Y576
EELWKHIQKELVDPSGLSEEQLKEIPyTK
SEQ ID NO. 80





82
HOOK2
NP_037444.1
Cytoskeletal protein
Y603
yVDKARMVMQTMEPK
SEQ ID NO: 81





83
KRT12
NP_000214.1
Cytoskeletal protein
Y262
TDLEMQIESLNEELAyMK
SEQ ID NO: 82





84
KRT20
NP_061883.1
Cytoskeletal protein
Y384
TTEyQLSTLEER
SEQ ID NO: 83





85
KRT2A
NP_000414.2
Cytoskeletal protein
Y268
yEDEINKRTAAENDFVTLK
SEQ ID NO: 84





86
KRTHB2
NP_149022.3
Cytoskeletal protein
Y451
GAFLyEPCGVSTPVLSTGVLR
SEQ ID NO: 85





87
SMTN
NP_599031.1
Cytoskeletal protein
Y896
EPDWKCVYTyIQEFYR
SEQ ID NO: 86





88
SMTN
NP_599031.1
Cytoskeletal protein
Y901
EPDWKCVYTYIQEFyR
SEQ ID NO: 87





89
SPTA1
NP_003117.1
Cytoskeletal protein
Y2304
GLNyYLPMVEEDEHEPKFEK
SEQ ID NO: 88





90
SPTBN2
NP_008877.1
Cytoskeletal protein
Y604
EyRPCDPQLVSERVAK
SEQ ID NO: 89





91
SPTBN4
NP_066022.1
Cytoskeletal protein
Y2457
SWVSLYCVLSKGELGFyKDSK
SEQ ID NO: 90





92
TUBA3
NP_006000.2
Cytoskeletal protein
Y432
EDMAALEKDyEEVGVDSVEGEGEEEGE
SEQ ID NO: 91







EY





93
TUBA6
NP_116093.1
Cytoskeletal protein
Y449
DYEEVGADSADGEDEGEEy
SEQ ID NO: 92





94
PXN

Cytoskeletal protein,
Y76
yAHQQPPSPLPVYSSSAK
SEQ ID NO: 93





Apoptosis





95
FLJ11806
NP_079100.2
DNA binding protein
Y273
LCEPEVLNSLEETySPFFR
SEQ ID NO: 94





96
SMARCA5
NP_003592.2
DNA binding protein
Y719
LSKMGESSLRNFTMDTESSVYNFEGEDyR
SEQ ID NO: 95





97
SON
NP_115571.1
DNA binding protein
Y909
LGQDPyRLGHDPYR
SEQ ID NO: 96





98
ZBED1
NP_004720.1
DNA binding protein
Y479
EVIAKELSKTYQETPEIDMFLNVATFLDP
SEQ ID NO: 97







RyK





99
CRY1
NP_004066.1
DNA binding protein;
Y266
LFyFKLTDLYKKVK
SEQ ID NO: 98





Lyase





100
ERCC6
NP_000115.1
DNA repair
Y1279
HDAIMDGASPDyVLVEAEANRVAQDALK
SEQ ID NO: 99





101
POLI
NP_009126.1
DNA repair
Y377
LGTGNyDVMTPMVDILMK
SEQ ID NO: 100





102
MCM4
NP_005905.2
DNA replication
Y730
IGSSRGMVSAyPR
SEQ ID NO: 101





103
POLA
NP_058633.2
DNA replication
Y1430
QFFTPKVLQDyR
SEQ ID NO: 102





104
SMC5L1
NP_055925.1
DNA replication
Y626
YWKTSFySNK
SEQ ID NO: 103





105
CTPS
NP_001896.1
Enzyme, misc.
Y473
LYGDADyLEER
SEQ ID NO: 104





106
DPYD
NP_000101.1
Enzyme, misc.
Y882
IAELMDKKLPSFGPyLEQRKK
SEQ ID NO: 105





107
ENTPD1
NP_001767.3
Enzyme, misc.
Y287
DPCFHPGyKKVVNVSDLYKTPCTK
SEQ ID NO: 106





108
GLCE
NP_056369.1
Enzyme, misc.
Y477
DHIFLNSALRATAPyK
SEQ ID NO: 107





109
GLULD1
NP_057655.1
Enzyme, misc.
Y490
yELENEEIAAERNK
SEQ ID NO: 108





110
GPAA1
NP_003792.1
Enzyme, misc.
Y328
VEALTLRGINSFRQyKYDLVAVGKALEG
SEQ ID NO: 109







MFR





111
GPAA1
NP_003792.1
Enzyme, misc.
Y330
VEALTLRGINSFRQYKyDLVAVGKALEG
SEQ ID NO: 110







MFR





112
NAGLU
NP_000254.2
Enzyme, misc.
Y92
VRGSTGVAMAGLHRyLR
SEQ ID NO: 111





113
PYGM
NP_005600.1
Enzyme, misc.
Y473
DFyELEPHKFQNKTNGITPR
SEQ ID NO: 112





114
TKTL1
NP_036385.2
Enzyme, misc.
Y112
RLSFVDVATGWLGQGLGVACGMAYTGK







yFDR
SEQ ID NO: 113





115
UMPS
NP_000364.1
Enzyme, misc.
Y37
SGLSSPIyIDLR
SEQ ID NO: 114





116
VARS
NP_006286.1
Enzyme, misc.
Y469
LHEEGIIyR
SEQ ID NO: 115





117
COL11A1
NP 542196.2
Extracellular matrix
Y329
AKLGVKANIVDDFQEYNYGTMESyQTEA
SEQ ID NO: 116







PR





118
COL16A1
NP_001847.3
Extracellular matrix
Y1108
GERGyTGSAGEKGEPGPPGSEGLPGPP
SEQ ID NO: 117







GPAGPRGER





119
FRAS1
NP_079350.4
Extracellular matrix
Y2722
GDASSIVSAICYTVPKSAMGSSLyALESG
SEQ ID NO: 118







SDFKSR





120
TLL2
NP_036597.1
Extracellular matrix
Y541
DGPTEESALIGHFCGyEK
SEQ ID NO: 119





121
TNXB
NP_061978.5
Extracellular matrix
Y1183
WTVPEGEFDSFVIQyKDR
SEQ ID NO: 120





122
GDI2
NP_001485.2
G protein regulator,
Y333
KSDIyVCMISFAHNVAAQGK
SEQ ID NO: 121





misc.





123
GDI2
NP_001485.2
G protein regulator,
Y442
MKRKKNDIyGED
SEQ ID NO: 122





misc.





124
DDEF2
NP_003878.1
GTPase activaing
Y763
AFMPSILQNETyGALLSGSPPPAQPAAP
SEQ ID NO: 123





protein, ARF

STTSAPPLPPR





125
RICS
NP_055530.2
GTPase activating
Y1208
VEyVSSLSSSVR
SEQ ID NO: 124





protein, Rac/Rho





126
RICS
NP_055530.2
GTPase activating
Y1557
QFCESKNGPPYPQGAGQLDyGSK
SEQ ID NO: 125





protein, Rac/Rho





127
RICS
NP_055530.2
GTPase activating
Y1680
QSSVTWSQYDNLEDyHSLPQHQR
SEQ ID NO: 126





protein, Rac/Rho





128
NF1
NP_000258.1
GTPase activaing
Y2556
RVAETDyEMETQR
SEQ ID NO: 127





protein, Ras





129
RALGPS2
NP_689876.2
Guanine nucleotide
Y420
NRLyHSLGPVTR
SEQ ID NO: 128





exchange factor, Ras





130
RASGRP3
NP_733772.1
Guanine nucleotide
Y523
QGyKCKDCGANCHKQCKDLLVLACR
SEQ ID NO: 129





exchange factor, Ras





131
DDX6
NP_004388.1
Helicase
Y462
SLYVAEyHSEPVEDEKP
SEQ ID NO: 130





132
NAV2
NP_660093.2
Helicase
Y1179
KSSMDGAQNQDDGyLALSSR
SEQ ID NO: 131





133
NAV2
NP_660093.2
Helicase
Y1579
THSLSNADGQYDPyTDSRFR
SEQ ID NO: 132





134
THEA
NP_056362.1
Hydrolase, esterase
Y364
YREASARKKIRLDRKyIVSCK
SEQ ID NO: 133





135
LEMD3
NP_055134.2
Inhibitor protein
Y667
EEEETRQMyDMWKLIDVLR
SEQ ID NO: 134





136
MIG-6
NP_061821.1
Inhibitor protein
Y341
SLPSyLNGVMPPTQSFAPDPK
SEQ ID NO: 135





137
MIG-6
NP_061821.1
Inhibitor protein
Y358
SLPSYLNGVMPPTQSFAPDPKyVSSK
SEQ ID NO: 136





138
HK2
NP_000180.2
Kinase (non-protein)
Y301
TEFDQEIDMGSLNPGKQLFEKMISGMyM
SEQ ID NO: 137







GELVR





139
PIK3CB
NP_006210.1
Kinase, lipid
Y436
TINPSKYQTIRKAGKVHyPVAWVNTMVF
SEQ ID NO: 138





DFK





140
PIK3CD
NP_005017.2
Kinase, lipid
Y440
CLyMWPSVPDEKGELLNPTGTVR
SEQ ID NO: 139





141
PIK4CA
NP_477352.1
Kinase, lipid
Y470
LYKYHSQyHTVAGNDIK
SEQ ID NO: 140





142
PIK4CA
NP_477352.1
Kinase, lipid
Y1096
NRYAGEVyGMIR
SEQ ID NO: 141





143
PIP5K1A
NP_003548.1
Kinase, lipid
Y470
GSSGNSCITyQPSVSGEHK
SEQ ID NO: 142





144
TTK
NP_003309.2
KINASE; Protein
Y374
LEETKEyQEPEVPESNQK
SEQ ID NO: 143





kinase, dual-specificity





145
LMTK2
NP_055731.2
KINASE; Protein
Y1468
STEQSWPHSAPySR
SEQ ID NO: 144





kinase, Ser/Thr





146
ILK
NP_00101479
KINASE; Protein
Y351
MyAPAWVAPEALQK
SEQ ID NO: 145




4.1
kinase, Ser/Thr (non-





receptor)





147
IRAK1
NP_001560.2
KINASE; Protein
Y395
TQTVRGTLAYLPEEyIKTGR
SEQ ID NO: 146





kinase, Ser/Thr (non-





receptor)





148
MAP4K5
NP_006566.2
KINASE; Protein
Y401
ISSyPEDNFPDEEK
SEQ ID NO: 147





kinase, Ser/Thr (non-





receptor)





149
NRK
NP_940867.1
KINASE; Protein
Y984
FVDDVNNNyYEAPSCPR
SEQ ID NO: 148





kinase, Ser/Thr (non-





receptor)





150
TLK1
NP_036422.3
KINASE; Protein
Y481
yAAVKIHQLNKSWRDEK
SEQ ID NO: 149





kinase, Ser/Thr (non-





receptor)





151
TTN
NP_003310.3
KINASE; Protein
Y1713
LRMINEFGyCSLDYGVAYSR
SEQ ID NO: 150





kinase, Ser/Thr (non-





receptor)





152
TTN
NP_003310.3
KINASE; Protein
Y1981
DESyEELLRKTK
SEQ ID NO: 151





kinase, Ser/Thr (non-





receptor)





153
KIAA2002
XP_940171.1
KINASE; Protein
Y387
EIEPNyESPSSNNQDKDSSQASK
SEQ ID NO: 152





kinase, Ser/Thr (non-





receptor, predicted)





154
KIAA2002
XP_940171.1
KINASE; Protein
Y531
SSAIRyQEVWTSSTSPR
SEQ ID NO: 153





kinase, Ser/Thr (non-





receptor, predicted)





155
KIAA2002
XP_940171.1
KINASE; Protein
Y635
NAIKVPIVINPNAyDNLAIYK
SEQ ID NO: 154





kinase, Ser/Thr (non-





receptor, predicted)





156
KIAA2002

KINASE; Protein
Y641
NAIKVPIVINPNAYDNLAIyK
SEQ ID NO: 155





kinase, Ser/Thr (non-





receptor, predicted)





157
KIAA2002
XP_940171.1
KINASE; Protein
Y665
TTSVISHTyEEIETESK
SEQ ID NO: 156





kinase, Ser/Thr (non-





receptor, predicted)





158
KIAA2002
XP_940171.1
KINASE; Protein
Y797
CSVEELyAIPPDADVAK
SEQ ID NO: 157





kinase, Ser/Thr (non-





receptor, predicted)





159
KIAA2002
XP_940171.1
KINASE; Protein
Y880
STSSPyHAGNLLQR
SEQ ID NO: 158





kinase, Ser/Thr (non





receptor, predicted)





160
TNK1
NP_003976.1
KINASE; Protein
Y661
ILEHYQWOLSAASRyVLARP
SEQ ID NO: 159





kinase, tyrosine (non-





receptor)





161
EPHA1
NP_005223.3
KINASE; Receptor
Y781
LLDDFDGTyETQGGK
SEQ ID NO: 160





tyrosine kinase





162
EPHB3
NP_004434.2
KINASE; Receptor
Y600
LQQyIAPGMK
SEQ ID NO: 161





tyrosine kinase





163
EPHB4
NP_004435.3
KINASE; Receptor
Y906
QPHySAFGSVGEWLR
SEQ ID NO: 162





tyrosine kinase





164
FLT1
NP_002010.1
KINASE; Receptor
Y1048
DIyKNPDYVR
SEQ ID NO: 163





tyrosine kinase





165
TIE1
NP_005415.1
KINASE; Receptor
Y969
QLLRFASDAANGMQyLSEKQFIHR
SEQ ID NO: 164





tyrosine kinase





166
PLEKHA5
NP_061885.2
Lipid binding protein
Y398
GGNRPNTGPLyTEADR
SEQ ID NO: 165





167
PRODH
NP_057419.2
Mitochondrial
Y412
PLIFNTyQCYLKDAYDNVTLDVELARR
SEQ ID NO: 166





168
PRSS15
NP_004784.2
Mitochondrial
Y394
yLLQEQLKIIK
SEQ ID NO: 167





169
SLC25A1
NP_005975.1
Mitochondrial
Y276
YRNTWDCGLQILKKEGLKAFyK
SEQ ID NO: 168





170
SLC25A5
NP_001143.1
Mitochondrial
Y191
AAYFGIyDTAK
SEQ ID NO: 169





171
TOP1MT
NP_443195.1
Mitochondrrial
Y455
ILSyNRANRWAILCNHQR
SEQ ID NO: 170





172
DNCH1
NP_001367.2
Motor protein
Y3379
KNYMSNPSYNyEIVNR
SEQ ID NO: 171





173
KIFlA
NP_004312.2
Motor protein
Y1666
DMHDWLyAFNPLLAGTIRSK
SEQ ID NO: 172





174
KIF2B
NP_115948.3
Motor protein
Y536
yANRVKKLNVDVR
SEQ ID NO: 173





175
MYH1
NP_005954.2
Motor protein
Y820
ESIFCIQyNVR
SEQ ID NO: 174





176
MYH10
NP_005955.1
Motor protein
Y285
TFHIFyQLLSGAGEHLK
SEQ ID NO: 175





177
MYH13
NP_003793.2
Motor protein
Y1351
HDCDLLREQyEEEQEAK
SEQ ID NO: 176





178
MYH2
NP_060004.2
Motor protein
Y413
ALCYPRVKVGNEyVTKGQTVEQVSNAV
SEQ ID NO: 177







GALAKAVYEK





179
MYH3
NP_002461.2
Motor protein
Y284
SyHIFYQILSNK
SEQ ID NO: 178





180
MYH3
NP_002461.2
Motor protein
Y288
SYHIFyQILSNK
SEQ ID NO: 179





181
MYH4
NP_060003.2
Motor protein
Y389
AAyLTSLNSADLLK
SEQ ID NO: 180





182
MYH8
NP_002463.1
Motor protein
Y1463
QKyEETQAELEASQK
SEQ ID NO: 181





183
MYH8
NP_002463.1
Motor protein
Y1855
ELTyQTEEDRK
SEQ ID NO: 182





184
MYO1D
NP_056009.1
Motor protein
Y885
HLyKMDPTKQYKVMKTIPLYNLTGLSVSN
SEQ ID NO: 183







GK





185
MYO1D
NP_056009.1
Motor protein
Y893
HLYKMDPTKQyKVMKTIPLYNLTGLSVSN
SEQ ID NO: 184







GK





186
MYO1D
NP_056009.1
Motor protein
Y902
HLYKMDPTKQYKVMKTIPLyNLTGLSVSN
SEQ ID NO: 185







GK





187
MYO1E
NP_004989.2
Motor protein
Y971
NQyVPYPHAPGSQR
SEQ ID NO: 186





188
MYO1E
NP_004989.2
Motor protein
Y989
SLyTSMARPPLPR
SEQ ID NO: 187





189
MYO5A
NP_000250.1
Motor protein
Y834
yKIRRAATIVLQSYLR
SEQ ID NO: 188





190
MYO5B
XP_371116.4
Motor protein
Y1046
VEyLSDGFLEKNR
SEQ ID NO: 189





191
MYBPC2
NP_004524.2
Myosin binding protein
Y1003
HTSCTVSDLIVGNEYyFR
SEQ ID NO: 190





192
PPP2R5C
NP_002710.2
Phosphatase,
Y443
NPQyTVYSQASTMSIPVAMETDGPLFE
SEQ ID NO: 191





regulatory subunit

DVQMLRK





193
PHLPP
NP_919431.1
PHOSPHATASE;
Y1200
HYQLDQLPDyYDTPL
SEQ ID NO: 192





Protein phosphatase,





Ser/Thr (non-receptor)





194
PPP1CA
NP_00100870
PHOSPHATASE;
Y317
yGQFSGLNPGGRPITPPR
SEQ ID NO: 193




9.1
Protein phosphatase,





Ser/Thr (non-receptor)





195
PTPN11
NP_002825.3
PHOSPHATASE;
Y263
LLySRKEGQRQENKNK
SEQ ID NO: 194





Protein phosphatase,





tyrosine (non-receptor)





196
PTPRS
NP_570923.2
PHOSPHATASE;
Y205
yECVATNSAGVRYSSPANLYVRVR
SEQ ID NO: 195





Receptor protein





phosphatase, tyrosine





197
PTPRT
NP_008981.3
PHOSPHATASE;
Y345
TTTGTWAETHIVDSPNyK
SEQ ID NO: 196





Receptor protein





phosphatase, tyrosine





198
PTPRT
NP_008981.3
PHOSPHATASE;
Y1003
CVRyWPDDTEVYGDIK
SEQ ID NO: 197





Receptor protein





phosphatase, tyrosine





199
PTPRT
NP_008981.3
PHOSPHATASE;
Y1011
YWPDDTEVyGDIKVTLIETEPLAEYVIRTF
SEQ ID NO: 198





Receptor protein

TVQK





phosphatase, tyrosine





200
TPTE
NP_954870.2
PHOSPHATASE;
Y509
LyLPKNELDNLHKQK
SEQ ID NO: 199





Receptor protein





phosphatase, tyrosine





201
PDE6C
NP_006195.2
Phosphodiesterase
Y277
SYLNCERySIGLLDMTK
SEQ ID NO: 200





202
PLCG1
NP_002651.2
Phospholipase
Y977
CyRDMSSFPETK
SEQ ID NO: 201





203
CPD
NP_001295.2
Protease (non-
Y520
FANEyPNITRLYSLGKSVESR
SEQ ID NO: 202





proteasomal)





204
CPD
NP_001295.2
Protease (non-
Y1344
LRQHHDEyEDEIR
SEQ ID NO: 203





poroteasomal)





205
CPD
NP_001295.2
Protease (non-
Y1376
SLLSHEFQDETDTEEETLySSKH
SEQ ID NO: 204





proteasomal)





206
MMP15
NP_002419.1
Protease (non-
Y525
PISVWQGIPASPKGAFLSNDAAyTYFYKG
SEQ ID NO: 205





proteasomal)

TK





207
MMP15
NP_002419.1
Protease (non-
Y527
PISVWQG IPASPKGAFLSNDAAYTyFYKG
SEQ ID NO: 206





proteasomal)

TK





208
NAALADL2
NP_996898.1
Protease (non-
Y110
LQEESDYITHyTR
SEQ ID NO: 207





proteasomal)





209
SENP6
NP_056386.1
Protease (non-
Y781
yEPNPHYHENAVIQK
SEQ ID NO: 208





proteasomal)





210
YME1L1
NP_055078.1
Protease (non-
Y646
FGMSEKLGVMTySDTGK
SEQ ID NO: 209





proteasomal)





211
F2R
NP_001983.1
Receptor, GPCR
Y420
MDTCSSNLNNSIyK
SEQ ID NO: 210





212
GABBR1
NP_001461.1
Receptor, GPCR
Y776
KMNTWLGIFYGyK
SEQ ID NO: 211





213
LPHN2
NP_036434.1
Receptor, GPCR
Y1350
RSENEDIyYK
SEQ ID NO: 212





214
OR2D3
NP_00100468
Receptor, GPCR
Y294
ELDKMISVFyTAVTPMLNPIIYSLR
SEQ ID NO: 213




4.1





215
OR2D3
NP_00100468
Receptor, GPCR
Y306
ELDKMISVFYTAVTPMLNPIIySLR
SEQ ID NO: 214




4.1





216
OR7G1
NP_00100519
Receptor, GPCR
Y278
ITAVASVMyTVVPQMMNPFIYSLR
SEQ ID NO: 215




2.1





217

BAC45258.1
Receptor, GPCR
Y475
yLGIMKPLTYPMRQK
SEQ ID NO: 216





218
IGF2R
NP_000867.1
Receptor, misc.
Y1834
TySVGVCTFAVGPEQGGCKDGGVCLLS
SEQ ID NO: 217







GTKGASFGR





219
LRP1B
NP_061027.2
Receptor, misc.
Y1708
LyWTDGNTINMANMDGSNSKILFQNQK
SEQ ID NO: 218





220
LRP6
NP_002327.1
Receptor, misc.
Y1584
SQYLSAEENyESCPPSPYTER
SEQ ID NO: 219





221
NEO1
NP_002490.1
Receptor, misc.
Y548
AyAASPTSITVTWETPVSGNGEIQNYK
SEQ ID NO: 220





222
NEO1
NP_002490.1
Receptor, misc.
Y572
YAASPTSITVTWETPVSGNGEIQNyK
SEQ ID NO: 221





223
NRP1
NP_003864.3
Receptor, misc.
Y920
DKLNTQSTySEA
SEQ ID NO: 222





224
NRP2
NP_003863.2
Receptor, misc.
Y720
SPVCMEFQyQATGGRGVALQVVR
SEQ ID NO: 223





225
ODZ2
XP_047995.9
Receptor, misc.
Y1601
YYLAVDPVSGSLYVSDTNSRRIyRVK
SEQ ID NO: 224





226
ODZ3
XP_371717.3
Receptor, misc.
Y1479
HAVQTTLESATAIAVSYSGVLyITETDEKK
SEQ ID NO: 225





227
ODZ4

Receptor, misc.
Y2547
TWSYTYLEKAGVCLPASLALPyR
SEQ ID NO: 226





228
ODZ4
XP_166254.6
Receptor, misc.
Y3071
QILYTAYGEIyMDTNPNFQIIIGYHGGLYD
SEQ ID NO: 227







PLTK





229
PEAR1
XP_371320.3
Receptor, misc.
Y1251
DLPSLPGGPRESSyMEMK
SEQ ID NO: 228





230
PLXNA1
NP_115618.2
Receptor, misc.
Y1585
QTSAyNISNSSTFTK
SEQ ID NO: 229





231
PLXNC1
NP_005752.1
Receptor, misc.
Y1350
EMyLTKLLSTKVAIHSVLEK
SEQ ID NO: 230





232
PLXND1
NP_055918.1
Receptor, misc.
Y1642
KLNTLAHyKIPEGASLAMSLIDKK
SEQ ID NO: 231





233
SDC1
NP_00100694
Receptor, misc.
Y286
KKDEGSySLEEPK
SEQ ID NO: 232




7.1





234
SDC1
NP_00100694
Receptor, misc.
Y299
QANGGAyQKPTKQEEFYA
SEQ ID NO: 233




7.1





235
SDC3
NP_055469.2
Receptor, misc.
Y441
QASVTYQKPDKQEEFyA
SEQ ID NO: 234





236
SIGIRR
NP_068577.1
Receptor, misc.
Y395
SSEVDVSDLGSRNySAR
SEQ ID NO: 235





237
SLAMF6
NP_443163.1
Receptor, misc.
Y308
ENDTITIySTINHSK
SEQ ID NO: 236





238
TLR10
NP_00101738
Receptor, misc.
Y786
EMyELQTFTELNEESR
SEQ ID NO: 237




8.1





239
SLC20A2
NP_006740.1
Receptor, misc.;
Y354
DSGLyKDLLHK
SEQ ID NO: 238





Transporter, facilitator





240
2BP1
NP_665899.1
RNA binding protein
Y358
VyAADPYHHALAPAPTYGVGAMASIYR
SEQ ID NO: 239





241
28P1
NP_665899.1
RNA binding protein
Y363
VYAADPyHHALAPAPTYGVGAMASIYR
SEQ ID NO: 240





242
CASC3
NP_031385.2
RNA binding protein
Y313
HQGLGGTLPPRTFINRNAAGTGRMSAP
SEQ ID NO: 241







RNySR





243
CSTF2
NP_001316.1
RNA binding protein
Y115
SLGTGAPVIESPyGETISPEDAPESISK
SEQ ID NO: 242





244
CSTF3
NP_001317.1
RNA binding protein
Y71
FWKLyIEAEIKAKNYDKVEK
SEQ ID NO: 243





245
FXR1

RNA binding protein
Y477
DPDSNPySLLDNTESDQTADTDASESHH
SEQ ID NO: 244







STNR





246
GLE1L
NP_00100372
RNA binding protein
Y547
KCPYSVPFYPTFKEGMALEDyQRMLGY
SEQ ID NO: 245




2.1


QVKDSK





247
HNRPR
NP_005817.1
RNA binding protein
Y434
STAYEDyYYHPPPR
SEQ ID NO: 246





248
ILF3
NP_004507.2
RNA binding protein
Y355
PKNENPVDyTVQIPPSTTYAITPMKRPME
SEQ ID NO: 247







EDGEEK





249
ILF3
NP_004507.2
RNA binding protein
Y365
PKNENPVDYTVQIPPSTTyAITPMKRPME
SEQ ID NO: 248







EDGEEK





250
PABPCS
NP_543022.1
RNA binding protein
Y15
yLKAALYVGDLDPDVTEDMLYKK
SEQ ID NO: 249





251
RAE1
NP_00101588
RNA binding protein
Y274
SNGTNTSAPQDIyAVNGIAFHPVHGTLAT
SEQ ID NO: 250




5.1


VGSDGR





252
RBM14
NP_006319.1
RNA binding protein
Y645
LPDAHSDyARYSGSYNDYLR
SEQ ID NO: 251





253
RBM14
NP_006319.1
RNA binding protein
Y648
LPDAHSDYARySGSYNDYLR
SEQ ID NO: 252





254
R8M14
NP_006319.1
RNA binding protein
Y655
LPDAHSDYARYSGSYNDyLRAAQMHSG
SEQ ID NO: 253







QRRM





255
RBM3
NP_006734.1
RNA binding protein
Y118
YyDSRPGGYGYGYGRSR
SEQ ID NO: 254





256
SNRPB2
NP_003083.1
RNA binding protein
Y28
RSLyALFSQFGHVVDIVALKTMKMR
SEQ ID NO: 255





257
SYNCRIP
NP_006363.3
RNA binding protein
Y481
GGyEDPYYGYEDFQVGARGRGGRGAR
SEQ ID NO: 256







GAAPSR





258
C1QA
NP_057075.1
Secreted protein
Y84
GDQGEPGPSGNPGKVGyPGPSGPLGA
SEQ ID NO: 257







RGIPGIK





259
CHGB
NP_001810.1
Secreted protein
Y173
SQREDEEEEEGENyQKGER
SEQ ID NO: 258





260
CHGB
NP_001810.1
Secreted protein
Y362
GYPGVQAPEDLEWERyRGR
SEQ ID NO: 259





261
F8
NP_000123.1
Secreted protein
Y2124
FSSLYISQFIIMySLDGKKWQTYR
SEQ ID NO: 260





262
F8
NP_000123.1
Secreted protein
Y2134
FSSLYISQFIIMYSLDGKKWQTyR
SEQ ID NO: 261





263
SEMG1
NP_002998.1
Secreted protein
Y220
NSHQNKGHyQNVVEVREEHSSK
SEQ ID NO: 262





264
SERP1
NP_003003.3
Secreted protein
Y127
PIyPCRWLCEAVRDSCEPVMQFFGFYW
SEQ ID NO: 263







PEMLK





265
WNT4
NP110388.2
Secreted protein
Y80
NLEVMDSVRRGAQLAIEECQyQFR
SEQ ID NO: 264





266
BARX1
NP_067545.2
Transcription factor
Y161
LSTPDRIDLAESLGLSQLQVKTWyQN
SEQ ID NO: 265







RR





267
CREB5
NP878901.2
Transcription factor
Y3
MIyEESKMNLEQER
SEQ ID NO: 266





268
DCP1A
NP_060873.3
Transcription factor
Y64
SASPyHGFTIVNR
SEQ ID NO: 267





269
EGR1

Transcription factor
Y26
EMQLMSPLQISDPFGSFPHsPTMDNY
SEQ ID NO: 268







PK





270
GATA6
NP_005248.2
Transcription factor
Y310
EPGGYAAAGSGGAGGVSGGGSSLAAM
SEQ ID NO: 269







GGREPQySSLSAAR





271
GATA6
NP_005248.2
Transcription factor
Y409
RDGTGHyLCNACGLYSKMNGLSR
SEQ ID NO: 270





272
HIC1
NP_006488.2
Transcription factor
Y136
HGKyCHLRGGGGGGGGYAPYGR
SEQ ID NO: 271





273
HIC1
NP_006488.2
Transcription factor
Y149
HGKYCHLRGGGGGGGGyAPYGR
SEQ ID NO: 272





274
HIC1
NP_006488.2
Transcription factor
Y152
HGKYCHLRGGGGGGGGYAPyGR
SEQ ID NO: 273





275
LITAF
NP_004853.2
Transcription factor
Y23
TGPSSAPSAPPSyEET
SEQ ID NO: 274





276
MECT1
NP_056136.1
Transcription factor
Y133
RQADSCPyGTMYLSP
SEQ ID NO: 275





277
MLL
NP_005924.2
Transcription factor
Y2136
PPHSQTSGSCYyHVISKVPRIRTPSYSPT
SEQ ID NO: 276







QR





278
MLX
NP_733752.1
Transcription factor
Y215
KDVTALKIMKVNyEQIVK
SEQ ID NO: 277





279
MYOD1
NP_002469.2
Transcription factor
Y230
RNCYEGAyYNEAPSEPRPGK
SEQ ID NO: 278





280
NFATC1
NP_006153.2
Transcription factor
Y688
RKRSQyQRFTYLPANVPIIK
SEQ ID NO: 279





281
PBX2
NP_002577.2
Transcription factor
Y384
HSMGPGGyGDNLGGGQMYSPREMR
SEQ ID NO: 280





282
PHOX2A
NP_005160.2
Transcription factor
Y75
DHQPAPYSAVPyKFFPEPSGLHEKR
SEQ ID NO: 281





283
PITX2
NP_000316.2
Transcription factor
Y116
QRTHFTSQQLQELEATFQRNRyPDMS
SEQ ID NO: 282







TR





284
PRKCBP1
NP_036540.3
Transcription factor
Y369
SIFNSAMQEMEVyVENIRRK
SEQ ID NO: 283





285
R.AI1
NP_109590.3
Transcription factor
Y185
THSLHVQQPPPPQQPLAyPK
SEQ ID NO: 284





286
RFX4
NP_002911.2
Transcription factor
Y214
LGTLLPEFPNVKDLNLPASLPEEKVSTFI
SEQ ID NO: 285







MMyR





287
RUNX3
NP_004341.1
TranscripUon factor
Y280
MHYPGAMSAAFPySATPSGTSISSLSVA
SEQ ID NO: 286







GMPATSR





288
SOX7
NP113627.1
Transcription factor
Y109
LQHMQDyPNYKYR
SEQ ID NO: 287





289
SOX7
NP113627.1
Transcription factor
Y112
LQHMQDYPNyKYR
SEQ ID NO: 288





290
TBX1
NP_005983.1
Transcription factor
Y38
MHFSTVTRDMEAFTASSLSSLGAAGGFP
SEQ ID NO: 289







GAASPGADPyGPR





291
TBX5
NP_000183.2
Transcription factor
Y100
VTGLNPKTKyILLMDIVPADDHRYK
SEQ ID NO: 290





292
TBX5
NP_000183.2
Transcription factor
Y114
VTGLNPKTKYILLMDIVPADDHRyK
SEQ ID NO: 291





293
TCF12
NP_003196.1
Transcription factor
Y195
KVPPGLPSSVyAPSPNSDDFNR
SEQ ID NO: 292





294
ZNF267
NP_003405.2
Transcnption factor
Y615
ECGKAFSySSDVIQHR
SEQ ID NO: 293





295
GTF2E1
NP_005504.1
Transcription initiation
Y91
HNyYFINYR
SEQ ID NO: 294





complex





296
GTF2H1
NP_005307.1
Transcription initiation
Y516
QyLSTNLVSHIEEMLQTAYNK
SEQ ID NO: 295





complex





297
GTF2H1
NP_005307.1
Transcription initiation
Y533
QYLSTNLVSHIEEMLQTAyNK
SEQ ID NO: 296





complex





298
GTF3C5
NP_036219.1
Transcription initiation
Y305
VLLPFIAYYMITGPWRSLWIRFGyDPR
SEQ ID NO: 297





complex





299
POLR1B
NP_061887.2
Transcription initiation
Y136
GIIKQFLGyVPIMVKSK
SEQ ID NO: 298





complex





300
POLR1B
NP_061887.2
Transcription initiation
Y1118
FVAELAAMNIK
SEQ ID NO: 299





complex





301
POLR3C
NP_006459.3
Transcription initiation
Y396
QVEDFAMIPAKEAKDMLyKMLSENFMSL
SEQ ID NO: 300





complex

QEIPK





302
POLRMT
NP_005026.3
Transcription initiation
Y386
LLRDVYAKDGRVSyPK
SEQ ID NO: 301





complex





303
PTRF
NP_036364.2
Transcription initiation
Y156
VMIyQDEVK
SEQ ID NO: 302





complex





304
PTRF

Transcription initiation
Y308
KSFTPDHVVyAR
SEQ ID NO: 303





complex





305
ES
NP_001121.2
Transcription,
Y64
HYVMyYEMSYGLNIEMHKQAEIVKR
SEQ ID NO: 304





coactivator/corepressor





306
ES
NP_001121.2
Transcription,
Y69
HYVMYYEMSyGLNIEMHKQAEIVKR
SEQ ID NO: 305





coactivator/corepressor





307
NKRD12
NP_056023.2
Transcription,
Y1229
PPVEyDSDFMLESSESQMSFSQSPFLSI
SEQ ID NO: 306





coactivator/corepressor

K





308
BCOR
NP_060215.4
Transcription
Y1527
LLLSYGADPTLATySGRTIMK
SEQ ID NO: 307





coactivator/corepressor





309
BRD8
NP_006687.3
Transcription
Y167
LEEEEAEVKRKATDAAyQARQAVK
SEQ ID NO: 308





coactivator/corepressor





310
CXXC1
NP_055408.1
Transcription,
Y509
yESQTSFGSMYPTR
SEQ ID NO: 309





coactivator/corepressor





311
CXXC1
NP_055408.1
Transcription,
Y519
YESQTSFGSMyPTR
SEQ ID NO: 310





coactivator/corepressor





312
EP400
NP_056224.2
Transcription,
Y1432
LKASRLFQPVQyGQKPEGRTVAFPSTHP
SEQ ID NO: 311





coactivator/corepressor

PR





313
HSFY1
NP149099.2
Transcription,
Y175
LKFyYNPNFK
SEQ ID NO: 312





coactivator/corepressor





314
HSFY1
NP149099.2
Transcription,
Y176
LKFYyNPNFK
SEQ ID NO: 313





coactivator/corepressor





315
HSGT1
NP_009196.1
Transcription,
Y64
KPGKGGVPAHMFGVTK
SEQ ID NO: 314





coactivator/corepressor





316
JARID1A
NP_005047.2
Transcrption,
Y148
VGSRLGyLPGKGTGSLLK
SEQ ID NO: 315





coactivator/corepressor





317
MKL2
NP_054767.3
Transcription
Y305
yHQYIPPDQKGEKNEPQMDSNYAR
SEQ ID NO: 316





coactivator/corepressor





318
MTA1
NP_004680.1
Transcription
Y659
MNWIDAPGDVFyMPK
SEQ ID NO: 317





coactivator/corepressor





319
PQBP1
NP_005701.1
Transcription,
Y187
REELAPyPK
SEQ ID NO: 318





coactivator/corepressor





320
PQBP1
NP_005701.1
Transcription,
Y209
VSRKDEELDPMDPSSySDAPR
SEQ ID NO: 319





coactivator/corepressor





321
PR1C285
NP_208384.2
Transcription
Y1845
yHEDAHMLDTQYRMHEGICAFPSVAFYK
SEQ ID NO: 320





coactivator/corepressor

SKLK





322
PR10285
NP_208384.2
Transcription,
Y1871
YHEDAHMLDTQYRMHEGICAFPSVAFyK
SEQ ID NO: 321





coactivator/corepressor

SKLK





323
TBL1XR1
NP_078941.2
Transcription
Y446
HQEPVySVAFSPDGR
SEQ ID NO: 322





coactivator/corepressor





324
THRAP3
NP_005110.1
Transcription
Y412
PFRGSQSPKRyKLR
SEQ ID NO: 323





coactivator/corepressor





325
TNIP1
NP_006049.2
Transcription
Y7
GPyRIYDPGGSVPSGEASAAFER
SEQ ID NO: 324





coactivator/corepressor





326
TNIP1
NP_006049.2
Transcription,
Y10
GPYRIyDPGGSVPSGEASAAFER
SEQ ID NO: 325





coactivator/corepressor





327
TP53BP2

Transcription,
Y541
QQHPENIySNSQGKP
SEQ ID NO: 326





coactivator/corepressor





328
YAP1
NP_006097.1
Transcription,
Y188
yFLNHIDQTTTWQDPR
SEQ ID NO: 327





coactivator/corepressor





329
ZBTB33
NP_006768.1
Transcription,
Y493
HDDHYELIVDGRVyYICIVCKRSYVCLTS
SEQ ID NO: 328





coactivator/corepressor

LR





330
ZBTB33
NP_006768.1
Transcription,
Y503
HDDHYELIVDGRVYYICIVCKRSyVCLTS
SEQ ID NO: 329





coactivator/corepressor

LR





331
B3GALT3
NP_003772.1
Transferase
Y175
yVMKTDTDVFINTGNLVK
SEQ ID NO: 330





332
CHST7
NP_063939.2
Transferase
Y414
GAAyGADRPFHLSARDAREAVHAWR
SEQ ID NO: 331





333
EXT1
NP_000118.2
Transferase
Y284
NALyHVHNGEDVVLLTTCK
SEQ ID NO: 332





334
F13A1

Transferase
Y482
LIVTKQIGGDGMMDITDTyK
SEQ ID NO: 333





335
GALGT
NP_001469.1
Transferase
Y504
yRYPGSLDESQMAKHR
SEQ ID NO: 334





336
GALNT3
NP_004473.1
Transferase
Y1O1
QNIDAGERPCLQGyYTAAELK
SEQ ID NO: 335





337
GALNT3
NP_004473.1
Transferase
Y102
QNIDAGERPCLQGYyTAAELK
SEQ ID NO: 336





338
HRMT1L3
NP_005779.1
Transferase
Y387
IAFWDDVyGFK
SEQ ID NO: 337





339
MTR
NP_000245.1
Transferase
Y701
yPRPLNIIEGPLMNGMK
SEQ ID NO: 338





340
MTR
NP_000245.1
Transferase
Y988
PFFDVWQLRGKyPNR
SEQ ID NO: 339





341
NDST3
NP_004775.1
Transferase
Y489
HTIFYKEyPGGPKEL
SEQ ID NO: 340





342
POFUT1

Transferase
Y211
yMVWSDEMVK
SEQ ID NO: 341





343
POMT1
NP_009102.2
Transferase
Y581
YSSSPLEWVTLDTNIAyWLHPR
SEQ ID NO: 342





344
SOAT1
NP_003092.4
Transferase
Y312
SSTVPIPTVNQYLYFLFAPTLIYRDSyPRN
SEQ ID NO: 343







PTVR





345
ST8SIA1
NP_003025.1
Transferase
Y217
TFVDNMKIYNHSyIYMPAFSMK
SEQ ID NO: 344





346
SULT1C2
NP_006579.2
Transferase
Y200
ILYLFyEDMKKNPK
SEQ ID NO: 345





347
SULT4A1
NP_055166.1
Transferase
Y114
SHLPyRFLPSDLHNGDSKVIYMARNPK
SEQ ID NO: 346





348
SULT4A1
NP_055166.1
Transferase
Y130
SHLPYRFLPSDLHNGDSKVIyMARNPK
SEQ ID NO: 347





349
TPST1
NP_003587.1
Transferase
Y350
VyKGEFQLPDFLKEKPQTEQVE
SEQ ID NO: 348





350
UGT2B10
NP_001066.1
Transferase
Y192
PPSyVPVVMSKLSDQMTFMERVKNML
SEQ ID NO: 349





351
EEF1A2
NP_001949.1
Translation initiation
Y85
FETTKyYITIIDAPGHR
SEQ ID NO: 350





complex





352
EEF1E1
NP_004271.1
Translation initiation
Y107
VyLTGYNFTLADILLYYGLHR
SEQ ID NO: 351





complex





353
EEF1E1
NP_004271.1
Translation initiation
Y111
VYLTGyNFTLADILLYYGLHR
SEQ ID NO: 352





complex





354
EIF3S6IP
NP_057175.1
Translation initiation
Y17
SEAAYDPyAYPSDYD
SEQ ID NO: 353





complex





355
EIF3S6IP
NP_057175.1
Translation initiation
Y19
AAYDPYAyPSDYDMH
SEQ ID NO: 354





complex





356
EIF3S6IP
NP_057175.1
Translation initiation
Y539
DMIHIADTKVARRyGDFFIRQIHK
SEQ ID NO: 355





complex





357
EIF3S8
NP_003743.1
Translation initiation
Y913
QQQSQTAy
SEQ ID NO: 356





complex





358
EIF3S9
NP_003742.2
Translation initiation
Y339
ARWTETyVR
SEQ ID NO: 357





complex





359
EIF4B
NP_001408.2
Translation initiation
Y105
LPKSPPYTAFLGNLPyDVTEESIK
SEQ ID NO: 358





complex





360
RPL7A
NP_000963.1
Translation initiation
Y226
TNyNDRYDEIRRHWGGNVLGPKSVAR
SEQ ID NO: 359





complex





361
RPL7A
NP_000963.1
Translation initiation
Y230
TNYNDRyDEIRRHWGGNVLGPKSVAR
SEQ ID NO: 360





complex





362
RPS13

Translation initiation
Y38
KLTSDDVKEQIyKL
SEQ ID NO: 361





complex





363
RPS16
NP_001011.1
Translation initiation
Y82
GGGHVAQIyAIR
SEQ ID NO: 362





complex





364
RPS3
NP_000996.2
Translation initiation
Y120
ACyGVLR
SEQ ID NO: 363





complex





365
TAF15
NP_003478.1
Translation initiation
Y434
GGRGGDRGGYGGDRSGGGYGGDRSS
SEQ ID NO: 364





complex; RNA binding

GGGySGDR





protein





366
TAF15
NP_003478.1
Translation initiation
Y443
SSGGGYSGDRSGGGyGGDRSGGGYGG
SEQ ID NO: 365





complex; RNA binding

DRGGGYGGDR





protein





367
TAF15

Translation initiation
Y460
GGGyGGDRGGYGGKMGGRNDYRND
SEQ ID NO: 366





complex; RNA binding

QR





protein





368
TAF15

Translation initiation
Y491
GGGyGGDRGGYGGKMGGRNDYRND
SEQ ID NO: 367





complex; RNA binding

QR





protein





369
TAF15
NP_003478.1
Translation initiation
Y528
GGGyGGDRGGYGGKMGGRNDYRND
SEQ ID NO: 368





complex; RNA binding

QR





protein





370
TAF15
NP_003478.1
Translation initiation
Y538
GGYGGDRGGGSGyGGDR
SEQ ID NO: 369





complex; RNA binding





protein





371
6004
NP_005836.1
Transporter, ABC
Y617
DGKMVQKGTyTEFLKSGIDFGSLLK
SEQ ID NO: 370





372
BCD3
NP_002849.1
Transporter, active
Y261
LRRPIGKMTITEQKyEGEYRYVNSR
SEQ ID NO: 371





373
BCD3
NP_002849.1
Transporter, active
Y265
LRRPIGKMTITEQKYEGEyR
SEQ ID NO: 372





374
ATP1A1
NP_000692.2
Transporter, active
Y542
EQPLDEELKDAFQNAyLELGGLGER
SEQ ID NO: 373





375
Atp1a3
NP_689509.1
Transporter, active
Y548
VLGFCHyYLPEEQFPK
SEQ ID NO: 374





376
Atp1a3
NP_689509.1
Transporter, active
Y549
VLGFCHYyLPEEQFPK
SEQ ID NO: 375





377
ATP7B
NP_000044.2
Transporter, active
Y187
NQEAVITyQPYLIQP
SEQ ID NO: 376





378
ATP8B2
NP_065185.1
Transporter, active
Y1162
SGyAFSHQEGFGELIMSGKNMR
SEQ ID NO: 377





379
CDW92
NP_071392.2
Transporter, active
Y263
VLVWILTILVILGSLGGTGVLWWLyAK
SEQ ID NO: 378





380
CDW92
NP_071392.2
Transporter, active
Y617
YNDGSPGREFyMDKVLMEFVENSRKA
SEQ ID NO: 379







MK





381
SLC7A11
NP_055146.1
Transporter, active
Y15
GGyLQGNVNGR
SEQ ID NO: 380





382
HBA2
NP_000508.1
Transporter, facilitator
Y25
VGAHAGEyGAEALER
SEQ ID NO: 381





383
Hba-a1
NP_005328.2
Transporter, facilitator
Y25
IGGHGAEyGAEALER
SEQ ID NO: 382





384
MATP
NP_00101252
Transporter, facilitator
Y105
PyILTLGVMMLVGMALYLNGATWAALIA
SEQ ID NO: 383




7.1


NPR





385
SLC12A2
NP_001037.1
Transporter, facilitator
Y227
IDHyRHTAAQLGEK
SEQ ID NO: 384





386
SLC12A2
NP_001037.1
Transporter, facilitator
Y275
DAVVTyTAESK
SEQ ID NO: 385





387
SLC27A2
NP_003636.1
Transporter, facilitator
Y304
yNVTVIQYIGELLRYLCNSPQKPNDR
SEQ ID NO: 386





388
SLC27A2
NP_003636.1
Transporter, facilitator
Y311
YNVTVIQyIGELLRYLCNSPQKPNDR
SEQ ID NO: 387





389
SLC38A2
NP_061849.2
Transporter, facilitator
Y20
FSISPDEDSSSySSNSDFNYSYPTK
SEQ ID NO: 388





390
SLC38A2
NP_061849.2
Transporter, facilitator
Y28
FSISPDEDSSSYSSNSDFNySYPTK
SEQ ID NO: 389





391
SLC39A6
NP_036451.2
Transporter, facilitator
Y522
HAHPQEVyNEYVPRG
SEQ ID NO: 390





392
SLC6A15
NP_060527.2
Transporter, facilitator
Y99
NGGGAyLLPYLILLMVIGIPLFFLELSVGQ
SEQ ID NO: 391







RIR





393
SLC6A15
NP_060527.2
Transporter, facilitator
Y103
NGGGAYLLPyLILLMVIGIPLFFLELSVGQ
SEQ ID NO: 392







RIR





394
SLC9A1
NP_003038.2
Transporter, facilitator
Y366
PyVEANISHKSHTTIKYFLK
SEQ ID NO: 393





395
SLC9A1
NP_003038.2
Transporter, facilitator
Y381
PYVEANISHKSHTTIKyFLK
SEQ ID NO: 394





396
PC
NP_000029.2
Tumor suppressor
Y737
NLMANRPAKyKDANIMSPGSSLPSLHV
SEQ ID NO: 395







RK





397
LZTS1
NP_066300.1
Tumor suppressor
Y295
LQRSFEEKELASSLAEERPR
SEQ ID NO: 396





398
PHF3
NP_055968.1
Tumor suppressor
Y1291
EICVVRFTPVTEEDQISYTLLFAyFSSRKR
SEQ ID NO: 397





399
RB1
NP_000312.2
Tumor suppressor
Y239
LSPPMLLKEPyKTAVIPINGSPR
SEQ ID NO: 398





400
SLIT2
NP_004778.1
Tumor suppressor
Y1502
RKySFECTDGSSFVDEVEKWK
SEQ ID NO: 399





401
TES
NP_056456.1
Tumor suppressor
Y111
KNVSINTVTyEWAPPVQNQALAR
SEQ ID NO: 400





402
TP53
NP_000537.2
Tumor suppressor;
Y327
KKPLDGEyFTLQIR
SEQ ID NO: 401





Transcription factor;





Activator protein





403
COPS6
NP_006824.2
Ubiquitin conjugating
Y105
EYyYTKEEQFK
SEQ ID NO: 402





system





404
COPS6
NP_006824.2
Ubiquitin conjugating
Y106
EYYyTKEEQFK
SEQ ID NO: 403





system





405
CUL2
NP_003582.2
Ubiquitin conjugating
Y43
ATWNDRFSDIyALCVAYPEPLGER
SEQ ID NO: 404





system





406
CUL5
NP_003469.2
Ubiquitin conjugating
Y214
FyRTQAPSYLQQNGVQNYMK
SEQ ID NO: 405





system





407
CUL5
NP_003469.2
Ubiquitin conjugating
Y221
FYRTQAPSyLQQNGVQNYMK
SEQ ID NO: 406





system





408
CUL5
NP_003469.2
Ubiquitin conjugating
Y230
FYRTQAPSYLQQNGVQNyMK
SEQ ID NO: 407





system





409
HERC4
NP_071362.1
Ubiquitin conjugating
Y895
QEFVDAYVDyIFNKSVASLFDAFHAGFHK
SEQ ID NO: 408





system

VCGGK





410
MGRN1

Ubiquitin conjugating
Y411
AIPSAPLyEEITYSG
SEQ ID NO: 409





system





411
MGRN1

Ubiquitin conjugating
Y416
PLYEEITySGISDGL
SEQ ID NO: 410





system





412
NEDD4
NP_006145.1
Ubiquitin conjugating
Y43
VIAGIGLAKKDILGASDPVR
SEQ ID NO: 411





system





413
NEDD4
NP_006145.1
Ubiquitin conjugating
Y150
VKGYLRLKMTyLPK
SEQ ID NO: 412





system





414
NYREN18
NP_057202.2
Ubiquitin conjugating
Y126
IAETFGLQENyIK
SEQ ID NO: 413





system





415
TNFAIP3
NP_006281.1
Ubiquitin conjugating
Y111
TNGDGNCLMHATSQyMWGVQDTDLVL
SEQ ID NO: 414





system

RK





416
TRIAD3
NP_996994.1
Ubiquitin conjugating
Y370
NYyDLNVLCNFLLENPDYPK
SEQ ID NO: 415





system





417
TRIAD3
NP_996994.1
Ubiquitin conjugating
Y385
NYyDLNVLCNFLLENPDyPK
SEQ ID NO: 416





system





418
UBE2E1
NP_003332.1
Ubiquitin conjugating
Y77
ELADITLDPPPNCSAGPKGDNIyEWR
SEQ ID NO: 417





system





419
UBE2J1
NP_057105.2
Ubiquitin conjugating
Y5
yNLKSPAVKRLMK
SEQ ID NO: 418





system





420
USP10
NP_005144.1
Ubiquitin conjugating
Y503
DIRPGAAFEPTyIYRLLTVNKSSLSEK
SEQ ID NO: 419





system





421
USP10
NP_005144.1
Ubiquitin conjugating
Y505
DIRPGAAFEPTYIyRLLTVNKSSLSEK
SEQ ID NO: 420





system





422
ZA20D1
NP_064590.1
Ubiquitin conjugating
Y794
VADSYSNGyREPPEPDGWAGGLR
SEQ ID NO: 421





system





423
AP1M1
NP_115882.1
Vesicle protein
Y354
EyLMRAHFGLPSVEAEDK
SEQ ID NO: 422





424
CLTC
NP_004850.1
Vesicle protein
Y899
FLRENPyYDSR
SEQ ID NO: 423





425
DYSF
NP_003485.1
Vesicle protein
Y1157
CyMYQARDLAAMDKDSFSDPYAIVSFLH
SEQ ID NO: 424







QSQK





426
DYSE
NP_003485.1
Vesicle protein
Y1159
CYMyQARDLAAMDKDSFSDPYAIVSFLH
SEQ ID NO: 425







QSQK





427
DYSF
NP_003485.1
Vesicle protein
Y1176
CYMYQARDLAAMDKDSFSDPyAIVSFLH
SEQ ID NO: 426







QSQK





428
ENTH
NP_055481.1
Vesicle protein
Y21
VRELVDKATNWMNySEIESK
SEQ ID NO: 427





429
ENTH
NP_055481.1
Vesicle protein
Y159
NKDKyVGVSSDSVGGFR
SEQ ID NO: 428





430
GOLGA3
NP_005886.2
Vesicle protein
Y210
ASTLAMTKEySFLR
SEQ ID NO: 429





431
GOLGA4
NP_002069.2
Vesicle protein
Y2148
NVyATTVGTPYK
SEQ ID NO: 430





432
GOLGB1
NP_004478.1
Vesicle protein
Y3005
SSSSQTQPLKVQyQR
SEQ ID NO: 431





433
GOLPH4
NP_055313.1
Vesicle protein
Y673
GREEHyEEEEEEEEDGAAVAEK
SEQ ID NO: 432





434
SCAMP3

Vesicle protein
Y35
QyATLDVYNPFETR
SEQ ID NO: 433





435
SCAMP4
NP_524558.1
Vesicle protein
Y205
EAQyNNFSGNSLPEYPTVPSYPGSGQ
SEQ ID NO: 434







WP





436
SEC10L1
NP_006535.1
Vesicle protein
Y356
QTFLSKLIKSIFISYLENYIEVETGyLKSR
SEQ ID NO: 435





437
SEC3L1
NP_060731.2
Vesicle protein
Y403
YAKLMEWLKSTDYGKyEGLTK
SEQ ID NO: 436





438
SEC3L1
NP_060731.2
Vesicle protein
Y800
VAQGIREEEVSyQLAFNKQELR
SEQ ID NO: 437





439
SEC8L1
NP_068579.3
Vesicle protein
Y247
KFLDTSHySTAGSSSVR
SEQ ID NO: 438





440
SNX25
NP_114159.2
Vesicle protein
Y151
PVVELLSNPOyINQMLLAQLAYREQMNE
SEQ ID NO: 439







HHK





441
SNX9
NP_057308.1
Vesicle protein
Y219
ASSSSMKIPLNKFPGFAKPGTEQyLLAK
SEQ ID NO: 440





442
STX4A
NP_004595.2
Vesicle protein
Y251
NILSSADyVER
SEQ ID NO: 441





443
TSG101
NP_006283.1
Vesicle protein
Y390
KTAGLSDLy
SEQ ID NO: 442





444
VPS28
NP_057292.1
Vesicle protein
Y36
EKyDNMAELFAVVKTMQALEK
SEQ ID NO: 443









The short name for each protein in which a phosphorylation site has presently been identified is provided in Column A, and its SwissProt accession number (human) is provided Column B. The protein type/group into which each protein falls is provided in Column C. The identified tyrosine residue at which phosphorylation occurs in a given protein is identified in Column D, and the amino acid sequence of the phosphorylation site encompassing the tyrosine residue is provided in Column E (lower case y=the tyrosine (identified in Column D)) at which phosphorylation occurs. Table 1 above is identical to FIG. 2, except that the latter includes the disease and cell type(s) in which the particular phosphorylation site was identified (Columns F and G).


The identification of these 443 phosphorylation sites is described in more detail in Part A below and in Example 1.


DEFINITIONS

As used herein, the following terms have the meanings indicated:


“Antibody” or “antibodies” refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies. The term “does not bind” with respect to an antibody's binding to one phospho-form of a sequence means does not substantially react with as compared to the antibody's binding to the other phospho-form of the sequence for which the antibody is specific.


“Carcinoma-related signaling protein” means any protein (or poly-peptide derived therefrom) enumerated in Column A of Table 1/FIG. 2, which is disclosed herein as being phosphorylated in one or more human carcinoma cell line(s). Carcinoma-related signaling proteins may be protein kinases, or direct substrates of such kinases, or may be indirect substrates downstream of such kinases in signaling pathways. A Carcinoma-related signaling protein may also be phosphorylated in other cell lines (non-carcinomic) harboring activated kinase activity.


“Heavy-isotope labeled peptide” (used interchangeably with AQUA peptide) means a peptide comprising at least one heavy-isotope label, which is suitable for absolute quantification or detection of a protein as described in WO/03016861, “Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry” (Gygi et al.), further discussed below.


“Protein” is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein.


“Phosphorylatable amino acid” means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.


“Phosphorylatable peptide sequence” means a peptide sequence comprising a phosphorylatable amino acid.


“Phosphorylation site-specific antibody” means an antibody that specifically binds a phosphorylatable peptide sequence/epitope only when phosphorylated, or only when not phosphorylated, respectively. The term is used interchangeably with “phospho-specific” antibody.


A. Identification of Novel Carcinoma-Related Signaling Protein Phosphorylation Sites.

The nearly 443 novel Carcinoma-related signaling protein phosphorylation sites disclosed herein and listed in Table 1/FIG. 2 were discovered by employing the modified peptide isolation and characterization techniques described in “Immunoaffinity Isolation of Modified Peptides From Complex Mixtures,” U.S. Patent Publication No. 20030044848, Rush et al. (the teaching of which is hereby incorporated herein by reference, in its entirety) using cellular extracts from the human carcinoma derived cell lines and patient samples indicated in Column G of Table 1/FIG. 2. Exemplary cell lines used include sw480, 293T, 293T TNT-TAT Silac, 293TTS ATIC-ALK, CTV-1, JB, Karpas 299, MOLT15, MV4-11, SU-DHL1, H196, H1993, Calu-3, HCT116, A431, U118 MG, DMS 153, SCLC T1, MDA-MB-468 and H1703. The isolation and identification of phosphopeptides from these cell lines, using an immobilized general phosphotyrosine-specific antibody, is described in detail in Example 1 below. In addition to the nearly 443 previously unknown protein phosphorylation sites (tyrosine) discovered, many known phosphorylation sites were also identified (not described herein).


The immunoaffinity/mass spectrometric technique described in the '848 patent Publication (the “IAP” method)—and employed as described in detail in the Examples—is briefly summarized below.


The IAP method employed generally comprises the following steps: (a) a proteinaceous preparation (e.g. a digested cell extract) comprising phosphopeptides from two or more different proteins is obtained from an organism; (b) the preparation is contacted with at least one immobilized general phosphotyrosine-specific antibody; (c) at least one phosphopeptide specifically bound by the immobilized antibody in step (b) is isolated; and (d) the modified peptide isolated in step (c) is characterized by mass spectrometry (MS) and/or tandem mass spectrometry (MS-MS). Subsequently, (e) a search program (e.g. Sequest) may be utilized to substantially match the spectra obtained for the isolated, modified peptide during the characterization of step (d) with the spectra for a known peptide sequence. A quantification step employing, e.g. SILAC or AQUA, may also be employed to quantify isolated peptides in order to compare peptide levels in a sample to a baseline.


In the IAP method as employed herein, a general phosphotyrosine-specific monoclonal antibody (commercially available from Cell Signaling Technology, Inc., Beverly, Mass., Cat #9411 (p-Tyr-100)) was used in the immunoaffinity step to isolate the widest possible number of phospho-tyrosine containing peptides from the cell extracts. Extracts from the human carcinoma cell lines described above were employed.


As described in more detail in the Examples, lysates were prepared from these cells line and digested with trypsin after treatment with DTT and iodoacetamide to alkylate cysteine residues. Before the immunoaffinity step, peptides were pre-fractionated by reversed-phase solid phase extraction using Sep-Pak C18 columns to separate peptides from other cellular components. The solid phase extraction cartridges were eluted with varying steps of acetonitrile. Each lyophilized peptide fraction was redissolved in IAP buffer and treated with phosphotyrosine-specific antibody (P-Tyr-100, CST #9411) immobilized on protein Agarose. Immunoaffinity-purified peptides were eluted with 0.1% TFA and a portion of this fraction was concentrated with Stage or Zip tips and analyzed by LC-MS/MS, using a ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer. Peptides were eluted from a 10 cm×75 μm reversed-phase column with a 45-min linear gradient of acetonitrile. MS/MS spectra were evaluated using the program Sequest with the NCBI human protein database.


This revealed a total of nearly 443 novel tyrosine phosphorylation sites in signaling pathways affected by kinase activation or active in carcinoma cells. The identified phosphorylation sites and their parent proteins are enumerated in Table 1/FIG. 2. The tyrosine (human sequence) at which phosphorylation occurs is provided in Column D, and the peptide sequence encompassing the phosphorylatable tyrosine residue at the site is provided in Column E. FIG. 2 also shows the particular type of carcinoma (see Column G) and cell line(s) (see Column F) in which a particular phosphorylation site was discovered.


As a result of the discovery of these phosphorylation sites, phospho-specific antibodies and AQUA peptides for the detection of and quantification of these sites and their parent proteins may now be produced by standard methods, described below. These new reagents will prove highly useful in, e.g., studying the signaling pathways and events underlying the progression of carcinomas and the identification of new biomarkers and targets for diagnosis and treatment of such diseases.


B. Antibodies and Cell Lines

Isolated phosphorylation site-specific antibodies that specifically bind a Carcinoma-related signaling protein disclosed in Column A of Table 1 only when phosphorylated (or only when not phosphorylated) at the corresponding amino acid and phosphorylation site listed in Columns D and E of Table 1/FIG. 2 may now be produced by standard antibody production methods, such as anti-peptide antibody methods, using the phosphorylation site sequence information provided in Column E of Table 1. For example, previously unknown Ser/Thr kinase phosphorylation site (tyrosine 351) (see Row 146 of Table 1/FIG. 2) is presently disclosed. Thus, antibodies that specifically bind this novel Ser/Thr kinase site can now be produced, e.g. by immunizing an animal with a peptide antigen comprising all or part of the amino acid sequence encompassing the respective phosphorylated residue (e.g. a peptide antigen comprising the sequence set forth in Rows 146 of Column E, of Table 1 (SEQ ID NO: 145) (which encompasses the phosphorylated tyrosine at positions 351 of the Ser/Thr kinase), to produce an antibody that only binds Ser/Thr kinase when phosphorylated at that site.


Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with a peptide antigen corresponding to the Carcinoma-related phosphorylation site of interest (i.e. a phosphorylation site enumerated in Column E of Table 1, which comprises the corresponding phosphorylatable amino acid listed in Column D of Table 1), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures. For example, a peptide antigen corresponding to all or part of the novel Receptor tyrosine kinase phosphorylation site disclosed herein (SEQ ID NO: 19=DDGMEEVVGHTQGPLDGSLyAK, encompassing phosphorylated tyrosine 365 (lowercase y; see Row 20 of Table 1)) may be used to produce antibodies that only bind Receptor tyrosine kinase phosphorylation when phosphorylated at tyr365. Similarly, a peptide comprising all or part of any one of the phosphorylation site sequences provided in Column E of Table 1 may employed as an antigen to produce an antibody that only binds the corresponding protein listed in Column A of Table 1 when phosphorylated (or when not phosphorylated) at the corresponding residue listed in Column D. If an antibody that only binds the protein when phosphorylated at the disclosed site is desired, the peptide antigen includes the phosphorylated form of the amino acid. Conversely, if an antibody that only binds the protein when not phosphorylated at the disclosed site is desired, the peptide antigen includes the non-phosphorylated form of the amino acid.


Peptide antigens suitable for producing antibodies of the invention may be designed, constructed and employed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)).


It will be appreciated by those of skill in the art that longer or shorter phosphopeptide antigens may be employed. See Id. For example, a peptide antigen may comprise the full sequence disclosed in Column E of Table 1/FIG. 2, or it may comprise additional amino acids flanking such disclosed sequence, or may comprise of only a portion of the disclosed sequence immediately flanking the phosphorylatable amino acid (indicated in Column E by lowercase “y”). Typically, a desirable peptide antigen will comprise four or more amino acids flanking each side of the phosphorylatable amino acid and encompassing it. Polyclonal antibodies produced as described herein may be screened as further described below.


Monoclonal antibodies of the invention may be produced in a hybridoma cell line according to the well-known technique of Kohler and Milstein. See Nature 265:495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of diagnostic assay methods provided by the invention. For example, a solution containing the appropriate antigen may be injected into a mouse or other species and, after a sufficient time (in keeping with conventional techniques), the animal is sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. Rabbit fusion hybridomas, for example, may be produced as described in U.S. Pat. No. 5,675,063, C. Knight, Issued Oct. 7, 1997. The hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.


Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246:1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)).


The preferred epitope of a phosphorylation-site specific antibody of the invention is a peptide fragment consisting essentially of about 8 to 17 amino acids including the phosphorylatable tyrosine, wherein about 3 to 8 amino acids are positioned on each side of the phosphorylatable tyrosine (for example, the OCLN tyrosine 315 phosphorylation site sequence disclosed in Row 44, Column E of Table 1), and antibodies of the invention thus specifically bind a target Carcinoma-related signaling polypeptide comprising such epitopic sequence. Particularly preferred epitopes bound by the antibodies of the invention comprise all or part of a phosphorylatable site sequence listed in Column E of Table 1, including the phosphorylatable amino acid.


Included in the scope of the invention are equivalent non-antibody molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho-specific manner, to essentially the same phosphorylatable epitope to which the phospho-specific antibodies of the invention bind. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.


Antibodies provided by the invention may be any type of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition fragments thereof. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,443,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.) The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)


The invention also provides immortalized cell lines that produce an antibody of the invention. For example, hybridoma clones, constructed as described above, that produce monoclonal antibodies to the Carcinoma-related signaling protein phosphorylation sties disclosed herein are also provided. Similarly, the invention includes recombinant cells producing an antibody of the invention, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)


Phosphorylation site-specific antibodies of the invention, whether polyclonal or monoclonal, may be screened for epitope and phospho-specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against the phospho and non-phospho peptide library by ELISA to ensure specificity for both the desired antigen (i.e. that epitope including a phosphorylation site sequence enumerated in Column E of Table 1) and for reactivity only with the phosphorylated (or non-phosphorylated) form of the antigen. Peptide competition assays may be carried out to confirm lack of reactivity with other phospho-epitopes on the given Carcinoma-related signaling protein. The antibodies may also be tested by Western blotting against cell preparations containing the signaling protein, e.g. cell lines over-expressing the target protein, to confirm reactivity with the desired phosphorylated epitope/target.


Specificity against the desired phosphorylated epitope may also be examined by constructing mutants lacking phosphorylatable residues at positions outside the desired epitope that are known to be phosphorylated, or by mutating the desired phospho-epitope and confirming lack of reactivity. Phosphorylation-site specific antibodies of the invention may exhibit some limited cross-reactivity to related epitopes in non-target proteins. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with non-target proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous to the Carcinoma-related signaling protein epitope for which the antibody of the invention is specific.


In certain cases, polyclonal antisera may exhibit some undesirable general cross-reactivity to phosphotyrosine itself, which may be removed by further purification of antisera, e.g. over a phosphotyramine column. Antibodies of the invention specifically bind their target protein (i.e. a protein listed in Column A of Table 1) only when phosphorylated (or only when not phosphorylated, as the case may be) at the site disclosed in corresponding Columns D/E, and do not (substantially) bind to the other form (as compared to the form for which the antibody is specific).


Antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to examine Carcinoma-related phosphorylation and activation status in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.


Antibodies may be further characterized by flow cytometry carried out according to standard methods. See Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: samples may be centrifuged on Ficoll gradients to remove erythrocytes, and cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37° C. followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary phosphorylation-site specific antibody of the invention (which detects a Carcinoma-related signal transduction protein enumerated in Table 1), washed and labeled with a fluorescent-labeled secondary antibody. Additional fluorochrome-conjugated marker antibodies (e.g. CD45, CD34) may also be added at this time to aid in the subsequent identification of specific hematopoietic cell types. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used.


Antibodies of the invention may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE) for use in multi-parametric analyses along with other signal transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell marker (CD34) antibodies.


Phosphorylation-site specific antibodies of the invention specifically bind to a human Carcinoma-related signal transduction protein or polypeptide only when phosphorylated at a disclosed site, but are not limited only to binding the human species, per se. The invention includes antibodies that also bind conserved and highly homologous or identical phosphorylation sites in respective Carcinoma-related proteins from other species (e.g. mouse, rat, monkey, yeast), in addition to binding the human phosphorylation site. Highly homologous or identical sites conserved in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human Carcinoma-related signal transduction protein phosphorylation sites disclosed herein.


C. Heavy-Isotope Labeled Peptides (AQUA Peptides).

The novel Carcinoma-related signaling protein phosphorylation sites disclosed herein now enable the production of corresponding heavy-isotope labeled peptides for the absolute quantification of such signaling proteins (both phosphorylated and not phosphorylated at a disclosed site) in biological samples. The production and use of AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861, “Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry,” Gygi et al., and also Gerber et al. Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which are hereby incorporated herein by reference, in their entirety).


The AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample. Briefly, the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and implementation using validated peptide internal standards to detect and quantify a target protein in sample. The method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.


Generally, to develop a suitable internal standard, a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest. The peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes (13C, 15N). The result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a mass shift. A newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision-induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.


The second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures. Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis. (See Gerber et al. supra.) AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above. The retention time and fragmentation pattern of the native peptide formed by digestion (e.g. trypsinization) is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures. Because an absolute amount of the AQUA peptide is added (e.g. 250 fmol), the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate. In addition, the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.


An AQUA peptide standard is developed for a known phosphorylation site sequence previously identified by the IAP-LC-MS/MS method within a target protein. One AQUA peptide incorporating the phosphorylated form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the non-phosphorylated form of the residue developed. In this way, the two standards may be used to detect and quantify both the phosphorylated and non-phosphorylated forms of the site in a biological sample.


Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage. Alternatively, a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced. Suitable proteases include, but are not limited to, serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.


A peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard. Preferably, the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins. Thus, a peptide is preferably at least about 6 amino acids. The size of the peptide is also optimized to maximize ionization frequency. Thus, peptides longer than about 20 amino acids are not preferred. The preferred ranged is about 7 to 15 amino acids. A peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.


A peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein. Alternatively, a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein. Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form). For example, peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.


The peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods. Preferably, the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragment masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids. As a result, the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum. Preferably, the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.


The label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice. The label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive. The label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as 13C, 15N, 17O, 18O, or 34S, are among preferred labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Preferred amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.


Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards. The internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas. The fragments are then analyzed, for example by multi-stage mass spectrometry (MSn) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature. Preferably, peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.


Fragment ions in the MS/MS and MS3 spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins. Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed. Generally, the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.


A known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate. The spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion. A separation is then performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample. Microcapillary LC is a preferred method.


Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MSn spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et al. supra.


In accordance with the present invention, AQUA internal peptide standards (heavy-isotope labeled peptides) may now be produced, as described above, for any of the nearly 443 novel Carcinoma-related signaling protein phosphorylation sites disclosed herein (see Table 1/FIG. 2). Peptide standards for a given phosphorylation site (e.g. the tyrosine 136 site in HIC1—see Row 272 of Table 1) may be produced for both the phosphorylated and non-phosphorylated forms of the site (e.g. see HIC1 site sequence in Column E, Row 272 of Table 1 (SEQ ID NO: 271)) and such standards employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.


AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column E of Table 1/FIG. 2). In a preferred embodiment, an AQUA peptide of the invention consists of, or comprises, a phosphorylation site sequence disclosed herein in Table 1/FIG. 2. For example, an AQUA peptide of the invention for detection/quantification of PIK3CB kinase when phosphorylated at tyrosine 436 may consist of, or comprise, the sequence TINPSKYQTIRKAGKVHyPVAWVNTMVFDFK (y=phosphotyrosine), which comprises phosphorylatable tyrosine 436 (see Row 139, Column E; (SEQ ID NO: 138)). Heavy-isotope labeled equivalents of the peptides enumerated in Table 1/FIG. 2 (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.


The phosphorylation site peptide sequences disclosed herein (see Column E of Table 1/FIG. 2) are particularly well suited for development of corresponding AQUA peptides, since the IAP method by which they were identified (see Part A above and Example 1) inherently confirmed that such peptides are in fact produced by enzymatic digestion (trypsinization) and are in fact suitably fractionated/ionized in MS/MS. Thus, heavy-isotope labeled equivalents of these peptides (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.


Accordingly, the invention provides heavy-isotope labeled peptides (AQUA peptides) for the detection and/or quantification of any of the Carcinoma-related phosphorylation sites disclosed in Table 1/FIG. 2 (see Column E) and/or their corresponding parent proteins/polypeptides (see Column A). A phosphopeptide sequence consisting of, or comprising, any of the phosphorylation sequences listed in Table 1 may be considered a preferred AQUA peptide of the invention. For example, an AQUA peptide comprising the sequence TQTVRGTLAYLPEEyIKTGR (SEQ ID NO: 146) (where y may be either phosphotyrosine or tyrosine, and where V=labeled valine (e.g. 14C)) is provided for the quantification of phosphorylated (or non-phosphorylated) kinase (Tyr 395) in a biological sample (see Row 147 of Table 1, tyrosine 395 being the phosphorylatable residue within the site). However, it will be appreciated that a larger AQUA peptide comprising a disclosed phosphorylation site sequence (and additional residues downstream or upstream of it) may also be constructed. Similarly, a smaller AQUA peptide comprising less than all of the residues of a disclosed phosphorylation site sequence (but still comprising the phosphorylatable residue enumerated in Column D of Table 1/FIG. 2) may alternatively be constructed. Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al. supra.).


Certain particularly preferred subsets of AQUA peptides provided by the invention are described above (corresponding to particular protein types/groups in Table 1, for example, Kinases or Adaptor/Scaffold proteins). Example 4 is provided to further illustrate the construction and use, by standard methods described above, of exemplary AQUA peptides provided by the invention. For example, the above-described AQUA peptides corresponding to the both the phosphorylated and non-phosphorylated forms of the disclosed PTPN11 phosphatase tyrosine 263 phosphorylation site (see Row 195 of Table 1/FIG. 2) may be used to quantify the amount of phosphorylated PTPN11 phosphatase (Tyr 263) in a biological sample, e.g. a tumor cell sample (or a sample before or after treatment with a test drug).


AQUA peptides of the invention may also be employed within a kit that comprises one or multiple AQUA peptide(s) provided herein (for the quantification of a Carcinoma-related signal transduction protein disclosed in Table 1/FIG. 2), and, optionally, a second detecting reagent conjugated to a detectable group. For example, a kit may include AQUA peptides for both the phosphorylated and non-phosphorylated form of a phosphorylation site disclosed herein. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.


AQUA peptides provided by the invention will be highly useful in the further study of signal transduction anomalies underlying cancer, including carcinomas, and in identifying diagnostic/bio-markers of these diseases, new potential drug targets, and/or in monitoring the effects of test compounds on Carcinoma-related signal transduction proteins and pathways.


D. Immunoassay Formats

Antibodies provided by the invention may be advantageously employed in a variety of standard immunological assays (the use of AQUA peptides provided by the invention is described separately above). Assays may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves a phosphorylation-site specific antibody of the invention), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.


In a heterogeneous assay approach, the reagents are usually the specimen, a phosphorylation-site specific antibody of the invention, and suitable means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.


Immunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al., “Methods for Modulating Ligand-Receptor Interactions and their Application”); U.S. Pat. No. 4,659,678 (Forrest et al., “Immunoassay of Antigens”); U.S. Pat. No. 4,376,110 (David et al., “Immunometric Assays Using Monoclonal Antibodies”). Conditions suitable for the formation of antigen-antibody complexes are well described. See id. Monoclonal antibodies of the invention may be used in a “two-site” or “sandwich” assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of a target Carcinoma-related signal transduction protein is detectable compared to background.


Phosphorylation site-specific antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies, or other target protein or target site-binding reagents, may likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.


Antibodies of the invention may also be optimized for use in a flow cytometry (FC) assay to determine the activation/phosphorylation status of a target Carcinoma-related signal transduction protein in patients before, during, and after treatment with a drug targeted at inhibiting phosphorylation at such a protein at the phosphorylation site disclosed herein. For example, bone marrow cells or peripheral blood cells from patients may be analyzed by flow cytometry for target Carcinoma-related signal transduction protein phosphorylation, as well as for markers identifying various hematopoietic cell types. In this manner, activation status of the malignant cells may be specifically characterized. Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 1% para-formaldehyde for 10 minutes at 37° C. followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary antibody (a phospho-specific antibody of the invention), washed and labeled with a fluorescent-labeled secondary antibody. Alternatively, the cells may be stained with a fluorescent-labeled primary antibody. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter EPICS-XL) according to the specific protocols of the instrument used. Such an analysis would identify the presence of activated Carcinoma-related signal transduction protein(s) in the malignant cells and reveal the drug response on the targeted protein.


Alternatively, antibodies of the invention may be employed in immunohistochemical (IHC) staining to detect differences in signal transduction or protein activity using normal and diseased tissues. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, supra. Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.


Antibodies of the invention may be also be optimized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN, Luminex™ and/or Bioplex™ assay formats, or otherwise optimized for antibody arrays formats, such as reversed-phase array applications (see, e.g. Paweletz et al., Oncogene 20(16): 1981-89 (2001)). Accordingly, in another embodiment, the invention provides a method for the multiplex detection of Carcinoma-related protein phosphorylation in a biological sample, the method comprising utilizing two or more antibodies or AQUA peptides of the invention to detect the presence of two or more phosphorylated Carcinoma-related signaling proteins enumerated in Column A of Table 1/FIG. 2. In one preferred embodiment, two to five antibodies or AQUA peptides of the invention are employed in the method. In another preferred embodiment, six to ten antibodies or AQUA peptides of the invention are employed, while in another preferred embodiment eleven to twenty such reagents are employed.


Antibodies and/or AQUA peptides of the invention may also be employed within a kit that comprises at least one phosphorylation site-specific antibody or AQUA peptide of the invention (which binds to or detects a Carcinoma-related signal transduction protein disclosed in Table 1/FIG. 2), and, optionally, a second antibody conjugated to a detectable group. In some embodies, the kit is suitable for multiplex assays and comprises two or more antibodies or AQUA peptides of the invention, and in some embodiments, comprises two to five, six to ten, or eleven to twenty reagents of the invention. The kit may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.


The following Examples are provided only to further illustrate the invention, and are not intended to limit its scope, except as provided in the claims appended hereto. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one of ordinary skill in the art.


EXAMPLE 1
Isolation of Phosphotyrosine-Containing Peptides from Extracts of Carcinoma Cell Lines and Identification of Novel Phosphorylation Sites

In order to discover previously unknown Carcinoma-related signal transduction protein phosphorylation sites, IAP isolation techniques were employed to identify phosphotyrosine-containing peptides in cell extracts from human carcinoma cell lines and patient cell lines identified in Column G of Table 1 including sw480, 293T, 293T TNT-TAT Silac, 293TTS ATIC-ALK, CTV-1, JB, Karpas 299, MOLT15, MV4-11, SU-DHL1, H196, H1993, Calu-3, HCT116, A431, U118 MG, DMS 153, SCLC T1, MDA-MB-468 and H1703. Tryptic phosphotyrosine-containing peptides were purified and analyzed from extracts of each of the cell lines mentioned above, as follows. Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin.


Suspension cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 mL lysis buffer per 1.25×108 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) and sonicated.


Adherent cells at about 80% confluency were starved in medium without serum overnight and stimulated, with ligand depending on the cell type or not stimulated. After complete aspiration of medium from the plates, cells were scraped off the plate in 10 ml lysis buffer per 2×108 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) and sonicated.


Sonicated cell lysates were cleared by centrifugation at 20,000×g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin, protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was added at 10-20 μg/mL. Digestion was performed for 1-2 days at room temperature.


Trifluoroacetic acid (TFA) was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak C18 columns (Waters) equilibrated with 0.1% TFA. A column volume of 0.7-1.0 ml was used per 2×108 cells. Columns were washed with 15 volumes of 0.1% TFA, followed by 4 volumes of 5% acetonitrile (MeCN) in 0.1% TFA. Peptide fraction I was obtained by eluting columns with 2 volumes each of 8, 12, and 15% MeCN in 0.1% TFA and combining the eluates. Fractions II and III were a combination of eluates after eluting columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA, respectively. All peptide fractions were lyophilized.


Peptides from each fraction corresponding to 2×108 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCl or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter (mainly in peptide fractions III) was removed by centrifugation. IAP was performed on each peptide fraction separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog number 9411) was coupled at 4 mg/ml beads to protein G (Roche), respectively. Immobilized antibody (15 μl, 60 μg) was added as 1:1 slurry in IAP buffer to 1 ml of each peptide fraction, and the mixture was incubated overnight at 4° C. with gentle rotation. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 75 μl of 0.1% TFA at room temperature for 10 minutes.


Alternatively, one single peptide fraction was obtained from Sep-Pak C18 columns by elution with 2 volumes each of 10%, 15%, 20%, 25%, 30%, 35% and 40% acetonitrile in 0.1% TFA and combination of all eluates. IAP on this peptide fraction was performed as follows: After lyophilization, peptide was dissolved in 50 ml IAP buffer (MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter was removed by centrifugation. Immobilized antibody (40 μl, 160 μg) was added as 1:1 slurry in IAP buffer, and the mixture was incubated overnight at 4° C. with gentle shaking. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 55 μl of 0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash of the beads (eluate 2) with 45 μl of 0.15% TFA. Both eluates were combined.


Analysis by LC-MS/MS Mass Spectrometry.

40 μl or more of IAP eluate were purified by 0.2 μl StageTips or ZipTips. Peptides were eluted from the microcolumns with 1 μl of 40% MeCN, 0.1% TFA (fractions I and II) or 1 μl of 60% MeCN, 0.1% TFA (fraction III) into 7.6-9.0 μl of 0.4% acetic acid/0.005% heptafluorobutyric acid. For single fraction analysis, 1 μl of 60% MeCN, 0.1% TFA, was used for elution from the microcolumns. This sample was loaded onto a 10 cm×75 μm PicoFrit capillary column (New Objective) packed with Magic C18 AQ reversed-phase resin (Michrom Bioresources) using a Famos autosampler with an inert sample injection valve (Dionex). The column was then developed with a 45-min linear gradient of acetonitrile delivered at 200 nl/min (Ultimate, Dionex), and tandem mass spectra were collected in a data-dependent manner with an LTQ ion trap mass spectrometer essentially as described by Gygi et al., supra.


Database Analysis & Assignments.

MS/MS spectra were evaluated using TurboSequest in the Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks 3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4×105; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The IonQuest and VuDta programs were not used to further select MS/MS spectra for Sequest analysis. MS/MS spectra were evaluated with the following TurboSequest parameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum number of differential amino acids per modification, 4; mass type parent, average; mass type fragment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis. Proteolytic enzyme was specified except for spectra collected from elastase digests.


Searches were performed against the NCBI human protein database (NCBI RefSeq protein release #11; 8 May 2005; 1,826,611 proteins, including 47,859 human proteins. Peptides that did not match RefSeq were compared to NCBI GenPept release #148; 15 Jun. 2005 release date; 2,479,172 proteins, including 196,054 human proteins.). Cysteine carboxamidomethylation was specified as a static modification, and phosphorylation was allowed as a variable modification on serine, threonine, and tyrosine residues or on tyrosine residues alone. It was determined that restricting phosphorylation to tyrosine residues had little effect on the number of phosphorylation sites assigned.


In proteomics research, it is desirable to validate protein identifications based solely on the observation of a single peptide in one experimental result, in order to indicate that the protein is, in fact, present in a sample. This has led to the development of statistical methods for validating peptide assignments, which are not yet universally accepted, and guidelines for the publication of protein and peptide identification results (see Carr et al., Mol. Cell Proteomics 3: 531-533 (2004)), which were followed in this Example. However, because the immunoaffinity strategy separates phosphorylated peptides from unphosphorylated peptides, observing just one phosphopeptide from a protein is a common result, since many phosphorylated proteins have only one tyrosine-phosphorylated site. For this reason, it is appropriate to use additional criteria to validate phosphopeptide assignments. Assignments are likely to be correct if any of these additional criteria are met: (i) the same sequence is assigned to co-eluting ions with different charge states, since the MS/MS spectrum changes markedly with charge state; (ii) the site is found in more than one peptide sequence context due to sequence overlaps from incomplete proteolysis or use of proteases other than trypsin; (iii) the site is found in more than one peptide sequence context due to homologous but not identical protein isoforms; (iv) the site is found in more than one peptide sequence context due to homologous but not identical proteins among species; and (v) sites validated by MS/MS analysis of synthetic phosphopeptides corresponding to assigned sequences, since the ion trap mass spectrometer produces highly reproducible MS/MS spectra. The last criterion is routinely employed to confirm novel site assignments of particular interest.


All spectra and all sequence assignments made by Sequest were imported into a relational database. The following Sequest scoring thresholds were used to select phosphopeptide assignments that are likely to be correct: RSp<6, XCorr≧2.2, and DeltaCN>0.099. Further, the sequence assignments could be accepted or rejected with respect to accuracy by using the following conservative, two-step process.


In the first step, a subset of high-scoring sequence assignments should be selected by filtering for XCorr values of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3 for +3, allowing a maximum RSp value of 10. Assignments in this subset should be rejected if any of the following criteria are satisfied: (i) the spectrum contains at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that can not be mapped to the assigned sequence as an a, b, or y ion, as an ion arising from neutral-loss of water or ammonia from a b or y ion, or as a multiply protonated ion; (ii) the spectrum does not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence is not observed at least five times in all the studies conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin).


In the second step, assignments with below-threshold scores should be accepted if the low-scoring spectrum shows a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference library-searching strategy.


EXAMPLE 2
Production of Phospho-Specific Polyclonal Antibodies for the Detection of Carcinoma-Related Signaling Protein Phosphorylation

Polyclonal antibodies that specifically bind a Carcinoma-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/FIG. 2) are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, as further described below. Production of exemplary polyclonal antibodies is provided below.


A. IRAK1 (Tyrosine 395).

A 20 amino acid phospho-peptide antigen, TQTVRGTLAYLPEEy*IKTGR (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 395 phosphorylation site in human IRAK kinase (see Row 147 of Table 1; SEQ ID NO: 146), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific IRAK1 (tyr 395) polyclonal antibodies as described in Immunization/Screening below.


B. TNS1 (Tyrosine 366).

A 20 amino acid phospho-peptide antigen, TQTVRGTLAYLPEEy*IKTGR (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 366 phosphorylation site in human SPRY1 (see Row 20 of Table 1 (SEQ ID NO: 19)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific TNS1 (tyr 366) polyclonal antibodies as described in Immunization/Screening below.


C. TBX1 (Tyrosine 38).

A 41 amino acid phospho-peptide antigen, MHFSTVTRDMEAFTASSLSSLGAAGGFPGAASPGADPy*GPR (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 38 phosphorylation site in human INPP5D protein (see Row 290 of Table 1 (SEQ ID NO: 289), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific TBX1 (tyr 38) antibodies as described in Immunization/Screening below.


Immunization/Screening.

A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and rabbits are injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (500 μg antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 μg antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affinity chromatography by standard methods (see ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.). The eluted immunoglobulins are further loaded onto a non-phosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the non-phosphorylated form of the phosphorylation site. The flow through fraction is collected and applied onto a phospho-synthetic peptide antigen-resin column to isolate antibodies that bind the phosphorylated form of the site. After washing the column extensively, the bound antibodies (i.e. antibodies that bind a phosphorylated peptide described in A-C above, but do not bind the non-phosphorylated form of the peptide) are eluted and kept in antibody storage buffer.


The isolated antibody is then tested for phospho-specificity using Western blot assay using an appropriate cell line that expresses (or overexpresses) target phospho-protein (i.e. phosphorylated IRAK1, TNS1 or TBX1), for example, DU145 or DMS79. Cells are cultured in DMEM or RPMI supplemented with 10% FCS. Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates is then measured. The loading buffer is added into cell lysate and the mixture is boiled at 100° C. for 5 minutes. 20 μl (10 μg protein) of sample is then added onto 7.5% SDS-PAGE gel.


A standard Western blot may be performed according to the Immunoblotting Protocol set out in the CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390. The isolated phospho-specific antibody is used at dilution 1:1000. Phosphorylation-site specificity of the antibody will be shown by binding of only the phosphorylated form of the target protein. Isolated phospho-specific polyclonal antibody does not (substantially) recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. TBX1 is not bound when not phosphorylated at tyrosine 38).


In order to confirm the specificity of the isolated antibody, different cell lysates containing various phosphorylated signal transduction proteins other than the target protein are prepared. The Western blot assay is performed again using these cell lysates. The phospho-specific polyclonal antibody isolated as described above is used (1:1000 dilution) to test reactivity with the different phosphorylated non-target proteins on Western blot membrane. The phospho-specific antibody does not significantly cross-react with other phosphorylated signal transduction proteins, although occasionally slight binding with a highly homologous phosphorylation-site on another protein may be observed. In such case the antibody may be further purified using affinity chromatography, or the specific immunoreactivity cloned by rabbit hybridoma technology.


EXAMPLE 3
Production of Phospho-Specific Monoclonal Antibodies for the Detection of Carcinoma-Related Signaling Protein Phosphorylation

Monoclonal antibodies that specifically bind a Carcinoma-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/FIG. 2) are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, and harvesting spleen cells from such animals to produce fusion hybridomas, as further described below. Production of exemplary monoclonal antibodies is provided below.


A. ILK (Tyrosine 351).

An 14 amino acid phospho-peptide antigen, My*APAWVAPEALQK (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 351 phosphorylation site in human ILK phosphatase (see Row 146 of Table 1 (SEQ ID NO: 145)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal ILK (tyr 351) antibodies as described in Immunization/Fusion/Screening below.


B. TP53BP2 (Tyrosine 541).

A 15 amino acid phospho-peptide antigen, QQHPENIy*SNSQGKP (where y*=phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 4505 phosphorylation site in human TP53BP2 (see Row 327 of Table 1 (SEQ ID NO: 326)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal TP53BP2 (tyr 541) antibodies as described in Immunization/Fusion/Screening below.


C. APC (Tyrosine 737).

A 29 amino acid phospho-peptide antigen, NLMANRPAKy*KDANIMSPGSSLPSLHVRK (where y*=phosphotyrosines) that corresponds to the sequence encompassing the tyrosine 737 phosphorylation site in human APC protein (see Row 396 of Table 1 (SEQ ID NO: 395)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal APC (tyr 737) antibodies as described in Immunization/Fusion/Screening below.


Immunization/Fusion/Screening.

A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and BALB/C mice are injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (e.g. 50 μg antigen per mouse). The mice are boosted with same antigen in incomplete Freund adjuvant (e.g. 25 μg antigen per mouse) every three weeks. After the fifth boost, the animals are sacrificed and spleens are harvested.


Harvested spleen cells are fused to SP2/0 mouse myeloma fusion partner cells according to the standard protocol of Kohler and Milstein (1975). Colonies originating from the fusion are screened by ELISA for reactivity to the phospho-peptide and non-phospho-peptide forms of the antigen and by Western blot analysis (as described in Example 1 above). Colonies found to be positive by ELISA to the phospho-peptide while negative to the non-phospho-peptide are further characterized by Western blot analysis. Colonies found to be positive by Western blot analysis are subcloned by limited dilution. Mouse ascites are produced from a single clone obtained from subcloning, and tested for phospho-specificity (against the ILK, TP53BP2, or APC) phospho-peptide antigen, as the case may be) on ELISA. Clones identified as positive on Western blot analysis using cell culture supernatant as having phospho-specificity, as indicated by a strong band in the induced lane and a weak band in the uninduced lane of the blot, are isolated and subcloned as clones producing monoclonal antibodies with the desired specificity.


Ascites fluid from isolated clones may be further tested by Western blot analysis. The ascites fluid should produce similar results on Western blot analysis as observed previously with the cell culture supernatant, indicating phospho-specificity against the phosphorylated target (e.g. ILK phosphorylated at tyrosine 351).


EXAMPLE 4
Production and Use of AQUA Peptides for the Quantification of Carcinoma-Related Signaling Protein Phosphorylation

Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of a Carcinoma-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/FIG. 2) are produced according to the standard AQUA methodology (see Gygi et al., Gerber et al., supra.) methods by first constructing a synthetic peptide standard corresponding to the phosphorylation site sequence and incorporating a heavy-isotope label. Subsequently, the MSn and LC-SRM signature of the peptide standard is validated, and the AQUA peptide is used to quantify native peptide in a biological sample, such as a digested cell extract. Production and use of exemplary AQUA peptides is provided below.


A. NF1 (Tyrosine 2556).

An AQUA peptide comprising the sequence, RVAETDy*EMETQR (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled valine (indicated by bold V), which corresponds to the tyrosine 2556 phosphorylation site in human PIK3C2B kinase (see Row 128 in Table 1 (SEQ ID NO: 127)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The Met (tyr 835) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated NF1 (tyr 2556) in the sample, as further described below in Analysis & Quantification.


B. TBX5 (Tyrosine 114).

An AQUA peptide comprising the sequence VTGLNPKTKYILLMDIVPADDHRy*K (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled proline (indicated by bold P), which corresponds to the tyrosine 114 phosphorylation site in human TBX5 protein (see Row 292 in Table 1 (SEQ ID NO: 291)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The TBX5 (tyr 114) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated TBX5 (tyr 114) in the sample, as further described below in Analysis & Quantification.


C. RB1 (Tyrosine 239).

An AQUA peptide comprising the sequence LSPPMLLKEPy*KTAVIPINGSPR (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled Leucine (indicated by bold L), which corresponds to the tyrosine 38 phosphorylation site in human VIM protein (see Row 399 in Table 1 (SEQ ID NO: 398)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The RB1 (tyr 239) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated RB1 (tyr 239) in the sample, as further described below in Analysis & Quantification.


D. MGRN1 (Tyrosine 416).

An AQUA peptide comprising the sequence PLYEEITySGISDGL (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled proline (indicated by bold P), which corresponds to the tyrosine 416 phosphorylation site in human MGRN1 protein (see Row 411 in Table 1 (SEQ ID NO: 410)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The MGRN1 (tyr 416) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated MGRN1 (tyr 416) in the sample, as further described below in Analysis & Quantification.


Synthesis & MS/MS Spectra.

Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, Calif.). Fmoc-derivatized stable-isotope monomers containing one 15N and five to nine 13C atoms may be obtained from Cambridge Isotope Laboratories (Andover, Mass.). Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 μmol. Amino acids are activated in situ with 1-H-benzotriazolium, 1-bis(dimethylamino)methylene]-hexafluorophosphate (1-),3-oxide:1-hydroxybenzotriazole hydrate and coupled at a 5-fold molar excess over peptide. Each coupling cycle is followed by capping with acetic anhydride to avoid accumulation of one-residue deletion peptide by-products. After synthesis peptide-resins are treated with a standard scavenger-containing trifluoroacetic acid (TFA)-water cleavage solution, and the peptides are precipitated by addition to cold ether. Peptides (i.e. a desired AQUA peptide described in A-D above) are purified by reversed-phase C18 HPLC using standard TFA/acetonitrile gradients and characterized by matrix-assisted laser desorption ionization-time of flight (Biflex III, Bruker Daltonics, Billerica, Mass.) and ion-trap (ThermoFinnigan, LCQ DecaXP) MS.


MS/MS spectra for each AQUA peptide should exhibit a strong y-type ion peak as the most intense fragment ion that is suitable for use in an SRM monitoring/analysis. Reverse-phase microcapillary columns (0.1 Ř150-220 mm) are prepared according to standard methods. An Agilent 1100 liquid chromatograph may be used to develop and deliver a solvent gradient [0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA)/7% methanol and 0.4% acetic acid/0.005% HFBA/65% methanol/35% acetonitrile] to the microcapillary column by means of a flow splitter. Samples are then directly loaded onto the microcapillary column by using a FAMOS inert capillary autosampler (LC Packings, San Francisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection.


Analysis & Quantification.

Target protein (e.g. a phosphorylated protein of A-D above) in a biological sample is quantified using a validated AQUA peptide (as described above). The IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.


LC-SRM of the entire sample is then carried out. MS/MS may be performed by using a ThermoFinnigan (San Jose, Calif.) mass spectrometer (LCQ DecaXP ion trap or TSQ Quantum triple quadrupole). On the DecaXP, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 150 ms per microscan, with two microscans per peptide averaged, and with an AGC setting of 1×108; on the Quantum, Q1 is kept at 0.4 and Q3 at 0.8 m/z with a scan time of 200 ms per peptide. On both instruments, analyte and internal standard are analyzed in alternation within a previously known reverse-phase retention window; well-resolved pairs of internal standard and analyte are analyzed in separate retention segments to improve duty cycle. Data are processed by integrating the appropriate peaks in an extracted ion chromatogram (60.15 m/z from the fragment monitored) for the native and internal standard, followed by calculation of the ratio of peak areas multiplied by the absolute amount of internal standard (e.g., 500 fmol).

Claims
  • 1. (canceled)
  • 2. (canceled)
  • 3. (canceled)
  • 4. (canceled)
  • 5. (canceled)
  • 6. (canceled)
  • 7. (canceled)
  • 8. (canceled)
  • 9. (canceled)
  • 10. (canceled)
  • 11. (canceled)
  • 12. (canceled)
  • 13. (canceled)
  • 14. (canceled)
  • 15. (canceled)
  • 16. An isolated phosphorylation site-specific antibody that specifically binds a human Carcinoma-related signaling protein selected from Column A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-443), wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.
  • 17. An isolated phosphorylation site-specific antibody that specifically binds a human Carcinoma-related signaling protein selected from Column A of Table 1 only when not phosphorylated at the tyrosine listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-443), wherein said antibody does not bind said signaling protein when phosphorylated at said tyrosine.
  • 18. (canceled)
  • 19. (canceled)
  • 20. (canceled)
  • 21. (canceled)
  • 22. (canceled)
  • 23. (canceled)
  • 24. (canceled)
  • 25. (canceled)
  • 26. (canceled)
  • 27. (canceled)
  • 28. (canceled)
  • 29. (canceled)
  • 30. (canceled)
  • 31. (canceled)
  • 32. (canceled)
  • 33. (canceled)
  • 34. (canceled)
  • 35. (canceled)
  • 36. (canceled)
  • 37. (canceled)
  • 38. (canceled)
  • 39. (canceled)
  • 40. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of an apoptosis protein selected from Column A, Rows 58-60, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 58-60, of Table 1 (SEQ ID NOs: 57-59), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 58-60 of Table 1.
  • 41. (canceled)
  • 42. (canceled)
  • 43. (canceled)
  • 44. (canceled)
  • 45. (canceled)
  • 46. (canceled)
  • 47. (canceled)
  • 48. (canceled)
  • 49. (canceled)
  • 50. (canceled)
  • 51. (canceled)
  • 52. (canceled)
  • 53. An isolated phosphorylation site-specific antibody according to claim 16, that specifically binds a human Leukemia-related signaling protein selected from Column A, Rows 442, 382, 34, 202, 424, 223, 161 and 43 of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 441, 381, 33, 201, 423, 222, 160 and 42), wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.
  • 54. An isolated phosphorylation site-specific antibody according to claim 17, that specifically binds a human Leukemia-related signaling protein selected from Column A, Rows 442, 382, 34, 202, 424, 223, 161 and 43 of Table 1 only when not phosphorylated at the tyrosine listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: SEQ ID NOs: 441, 381, 33, 201, 423, 222, 160 and 42), wherein said antibody does not bind said signaling protein when phosphorylated at said tyrosine.
  • 55. A method selected from the group consisting of: (a) a method for detecting a human leukemia-related signaling protein selected from Column A of Table 1, wherein said human leukemia-related signaling protein is phosphorylated at the tyrosine listed in corresponding Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-443), comprising the step of adding an isolated phosphorylation-specific antibody according to claim 16, to a sample comprising said human leukemia-related signaling protein under conditions that permit the binding of said antibody to said human leukemia-related signaling protein, and detecting bound antibody;(b) a method for quantifying the amount of a human leukemia-related signaling protein listed in Column A of Table 1 that is phosphorylated at the corresponding tyrosine listed in Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-443), in a sample using a heavy-isotope labeled peptide (AQUA™ peptide), said labeled peptide comprising a phosphorylated tyrosine at said corresponding tyrosine listed Column D of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 as an internal standard; and(c) a method comprising step (a) followed by step (b).
  • 56. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding STX4 only when phosphorylated at Y251, comprised within the phosphorylatable peptide sequence listed in Column E, Row 442, of Table 1 (SEQ ID NO: 442), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 57. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding STX4 only when not phosphorylated at Y251, comprised within the phosphorylatable peptide sequence listed in Column E, Row 442, of Table 1 (SEQ ID NO: 441), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 58. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding HBA1 only when phosphorylated at Y25, comprised within the phosphorylatable peptide sequence listed in Column E, Row 382, of Table 1 (SEQ ID NO: 381), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 59. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding HBA1 only when not phosphorylated at Y25, comprised within the phosphorylatable peptide sequence listed in Column E, Row 382, of Table 1 (SEQ ID NO: 381), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 60. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding F11R only when phosphorylated at Y280, comprised within the phosphorylatable peptide sequence listed in Column E, Row 34, of Table 1 (SEQ ID NO: 33), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 61. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding F11R only when not phosphorylated at Y280, comprised within the phosphorylatable peptide sequence listed in Column E, Row 34, of Table 1 (SEQ ID NO: 33), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 62. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding PLCG1 only when phosphorylated at Y977, comprised within the phosphorylatable peptide sequence listed in Column E, Row 202, of Table 1 (SEQ ID NO: 201), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 63. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding PLCG1 only when not phosphorylated at Y977, comprised within the phosphorylatable peptide sequence listed in Column E, Row 202, of Table 1 (SEQ ID NO: 201), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 64. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding CLTC only when phosphorylated at Y899, comprised within the phosphorylatable peptide sequence listed in Column E, Row 424, of Table 1 (SEQ ID NO: 423), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 65. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding CLTC only when not phosphorylated at Y899, comprised within the phosphorylatable peptide sequence listed in Column E, Row 424, of Table 1 (SEQ ID NO: 423), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 66. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding NRP1 only when phosphorylated at Y920, comprised within the phosphorylatable peptide sequence listed in Column E, Row 223, of Table 1 (SEQ ID NO: 222), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 67. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding NRP1 only when not phosphorylated at Y920, comprised within the phosphorylatable peptide sequence listed in Column E, Row 223, of Table 1 (SEQ ID NO: 222), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 68. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding EphA1 only when phosphorylated at Y781, comprised within the phosphorylatable peptide sequence listed in Column E, Row 1611, of Table 1 (SEQ ID NO: 160), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 69. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding EphA1 only when not phosphorylated at Y781, comprised within the phosphorylatable peptide sequence listed in Column E, Row 161, of Table 1 (SEQ ID NO: 160), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
  • 70. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding OCLN only when phosphorylated at Y287, comprised within the phosphorylatable peptide sequence listed in Column E, Row 43, of Table 1 (SEQ ID NO: 42), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 71. The method of claim 55, wherein said isolated phosphorylation-specific antibody is capable of specifically binding OCLN only when not phosphorylated at Y287, comprised within the phosphorylatable peptide sequence listed in Column E, Row 43, of Table 1 (SEQ ID NO: 42), wherein said antibody does not bind said protein when phosphorylated at said tyrosine.
RELATED APPLICATIONS

This application claims the benefit of, and priority to, PCT serial number PCT/US06/034063, filed Aug. 31, 2006, presently pending, the disclosure of which is incorporated herein, in its entirety, by reference.

Related Publications (1)
Number Date Country
20090061459 A1 Mar 2009 US
Provisional Applications (1)
Number Date Country
60712997 Aug 2005 US
Continuations (1)
Number Date Country
Parent PCT/US06/34063 Aug 2006 US
Child 12074228 US