Patent Publication Number: US-2010120055-A1

Title: Tyrosine phosphorylation sites

Description:
RELATED APPLICATIONS 
     Pursuant to 35 U.S.C. §119(e) this application claims the benefit of, and priority to, provisional application U.S. Ser. No. 60/847,749, filed Sep. 28, 2006, the disclosure of which is incorporated herein, in its entirety, by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention relates generally to novel tyrosine phosphorylation sites, methods and compositions for detecting, quantitating and modulating same. 
     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 to mention but a few: 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. (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 disease states 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. The identification of key signaling mechanisms is highly desirable in many contexts in addition to cancer. 
     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 underlie 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 ontogenesis in carcinoma by identifying the downstream signaling proteins mediating cellular transformation in these cancers. 
     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 diseases including for example, 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 many diseases. 
     SUMMARY OF THE INVENTION 
     The present invention provides in one aspect novel tyrosine phosphorylation sites (Table 1) identified in carcinoma. The novel sites occur in proteins such as: adaptor/scaffold proteins, adhesion/extra cellular matrix proteins, calcium binding proteins, cell cycle regulation proteins, chromatin or DNA binding/repair/replication proteins, chaperone proteins, cytoskeleton proteins, enzyme proteins, g proteins or regulator proteins, kinases (non-protein), ligand receptors, lipid binding proteins, protein kinases, mitochondrial proteins, motor or contractile proteins, proteases, phosphatases, receptor/channel/transporter/cell surface proteins, RNA binding proteins, secreted proteins, transcriptional regulators, tumor suppressor proteins, ubiquitan conjugating proteins, proteins of unknown function and vesicle proteins. 
     In another aspect, the invention provides peptides comprising the novel phosphorylation sites of the invention, and proteins and peptides that are mutated to eliminate the novel phosphorylation sites. 
     In another aspect, the invention provides modulators that modulate tyrosine phosphorylation at a novel phosphorylation site of the invention, including small molecules, peptides comprising a novel phosphorylation site, and binding molecules that specifically bind at a novel phosphorylation site, including but not limited to antibodies or antigen-binding fragments thereof. 
     In another aspect, the invention provides compositions for detecting, quantitating or modulating a novel phosphorylation site of the invention, including peptides comprising a novel phosphorylation site and antibodies or antigen-binding fragments thereof that specifically bind at a novel phosphorylation site. In certain embodiments, the compositions for detecting, quantitating or modulating a novel phosphorylation site of the invention are Heavy-Isotype Labeled Peptides (AQUA peptides) comprising a novel phosphorylation site. 
     In another aspect, the invention discloses phosphorylation site specific antibodies or antigen-binding fragments thereof. In one embodiment, the antibodies specifically bind to an amino acid sequence comprising a phosphorylation site identified in Table 1 when the tyrosine identified in Column D is phosphorylated, and do not significantly bind when the tyrosine is not phosphorylated. In another embodiment, the antibodies specifically bind to an amino acid sequence comprising a phosphorylation site when the tyrosine is not phosphorylated, and do not significantly bind when the tyrosine is phosphorylated. 
     In another aspect, the invention provides a method for making phosphorylation site-specific antibodies. 
     In another aspect, the invention provides compositions comprising a peptide, protein, or antibody of the invention, including pharmaceutical compositions. 
     In a further aspect, the invention provides methods of treating or preventing carcinoma in a subject, wherein the carcinoma is associated with the phosphorylation state of a novel phosphorylation site in Table 1, whether phosphorylated or dephosphorylated. In certain embodiments, the methods comprise administering to a subject a therapeutically effective amount of a peptide comprising a novel phosphorylation site of the invention. In certain embodiments, the methods comprise administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds at a novel phosphorylation site of the invention. 
     In a further aspect, the invention provides methods for detecting and quantitating phosphorylation at a novel tyrosine phosphorylation site of the invention. 
     In another aspect, the invention provides a method for identifying an agent that modulates tyrosine phosphorylation at a novel phosphorylation site of the invention, comprising: contacting a peptide or protein comprising a novel phosphorylation site of the invention with a candidate agent, and determining the phosphorylation state or level at the novel phosphorylation site. A change in the phosphorylation state or level at the specified tyrosine in the presence of the test agent, as compared to a control, indicates that the candidate agent potentially modulates tyrosine phosphorylation at a novel phosphorylation site of the invention. 
     In another aspect, the invention discloses immunoassays for binding, purifying, quantifying and otherwise generally detecting the phosphorylation of a protein or peptide at a novel phosphorylation site of the invention. 
     Also provided are pharmaceutical compositions and kits comprising one or more antibodies or peptides of the invention and methods of using them. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram depicting the immuno-affinity isolation and mass-spectrometric characterization methodology (IAP) used in the Examples to identify the novel phosphorylation sites disclosed herein. 
         FIG. 2  is a table (corresponding to Table 1) summarizing the 318 novel phosphorylation sites of the invention: Column A=the parent proteins from which the phosphorylation sites are derived; Column B=the SwissProt accession number for the human homologue of the identified parent proteins; Column C=the protein type/classification; Column D=the tyrosine residues at which phosphorylation occurs (each number refers to the amino acid residue position of the tyrosine in the parent human protein, according to the published sequence retrieved by the SwissProt accession number); Column E=flanking sequences of the phosphorylatable tyrosine residues; sequences (SEQ ID NOs: 1, 3-7, 9-46, 48, 50-60, 62-65, 67-73, 75-95, 97-168, 170-209, 211, 213-237, 239-240, 242-247, 249-268, 270-272, 274-279, 281-283, 285-295, 297, 299-308, 310-315, 317-325, 327-341) were identified using Trypsin digestion of the parent proteins; in each sequence, the tyrosine (see corresponding rows in Column D) appears in lowercase; Column F=the type of carcinoma in which each of the phosphorylation site was discovered; Column G=the cell type(s)/Tissue/Patient Sample in which each of the phosphorylation site was discovered; and Column H=the SEQ ID NOs of the trypsin-digested peptides identified in Column E. 
         FIG. 3  is an exemplary mass spectrograph depicting the detection of the phosphorylation of tyrosine 818 in DSC3, as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (corresponds to lowercase “y” in Column E of Table 1; SEQ ID NO: 19). 
         FIG. 4  is an exemplary mass spectrograph depicting the detection of the phosphorylation of tyrosine 499 in Plakophilin 1, as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (corresponds to lowercase “y” in Column E of Table 1; SEQ ID NO: 36). 
         FIG. 5  is an exemplary mass spectrograph depicting the detection of the phosphorylation of tyrosine 641 in DLL1, as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (corresponds to lowercase “y” in Column E of Table 1; SEQ ID NO: 17). 
         FIG. 6  is an exemplary mass spectrograph depicting the detection of the phosphorylation of tyrosine 52 in LRRC7, as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (corresponds to lowercase “y” in Column E of Table 1; SEQ ID NO: 29). 
         FIG. 7  is an exemplary mass spectrograph depicting the detection of the phosphorylation of tyrosine 104 in Plakophilin 1, as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* (and pY) indicates the phosphorylated tyrosine (corresponds to lowercase “y” in Column E of Table 1; SEQ ID NO: 35). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The inventors have discovered and disclosed herein novel tyrosine phosphorylation sites in signaling proteins extracted from carcinoma cells. The newly discovered phosphorylation sites significantly extend our knowledge of kinase substrates and of the proteins in which the novel sites occur. The disclosure herein of the novel phosphorylation sites and reagents including peptides and antibodies specific for the sites add important new tools for the elucidation of signaling pathways that are associate with a host of biological processes including cell division, growth, differentiation, developmental changes and disease. Their discovery in carcinoma cells provides and focuses further elucidation of the disease process. And, the novel sites provide additional diagnostic and therapeutic targets. 
     1. Novel Phosphorylation Sites in Carcinoma 
     In one aspect, the invention provides 318 novel tyrosine phosphorylation sites in signaling proteins from cellular extracts from a variety of human carcinoma-derived cell lines and tissue samples (such as H1993, lung HCC827, etc., as further described below in Examples), identified using the techniques described in “Immunoaffinity Isolation of Modified Peptides From Complex Mixtures,” U.S. Patent Publication No. 20030044848, Rush et al., using Table 1 summarizes the identified novel phosphorylation sites. 
     These phosphorylation sites thus occur in proteins found in carcinoma. The sequences of the human homologues are publicly available in SwissProt database and their Accession numbers listed in Column B of Table 1. The novel sites occur in proteins such as: g protein or regulator protein, adhesion or extra-cellular matrix proteins, cytoskeletal proteins, enzyme proteins, kinases (non-protein), motor or contractile proteins, protein kinases, receptor/channel/transporter/cell surface proteins, RNA binding proteins and transcriptional regulator proteins. (see Column C of Table 1). 
     The novel phosphorylation sites of the invention were identified according to the methods described by Rush et al., U.S. Patent Publication No. 20030044848, which are herein incorporated by reference in its entirety. Briefly, phosphorylation sites were isolated and characterized by immunoaffinity isolation and mass-spectrometric characterization (IAP) ( FIG. 1 ), using the following human carcinoma-derived cell lines and tissue samples: 23132/87; 293T; 293T(ATIC-ALK); 293T(NPM-ALK); 293T(NPM-ALKIATIC-ALK); 293T(ZNF198-FGFR); 3T3; 3T3(Abl); 3T3(Src); 42-MG-BA; 5637; 639L; 8-MG-BA; A 431; A 431: EGF, Iressa; A172; A498; A549; A704; AGS; ARH-77; AU-565; B13AML; B16AML; B17AML; B18AML; B20-XY1; B23-XY2; B24-XY2; B24_AML; B25-XY2; B25_AML; B29-XY2; B29_AML; B30-XY2; B32-XY2; B34-XY2; B36-XY2; B37-XY2; B38-XY2; B39-XY2; B41-XY2; BC-3C; BC001; BC002; BC003; BC004; BC005; BC007; BC008; BJ629; BJ630; BJ631; BJ635; BJ665; BJ669; BT-20; BT-549; BT1; BT2; C2C12-D; C2C12-D: Insulin; CAKI-2; CAL-29; CAL-51; CAL-85-1; CAMA-1; CAS-1; CCF-STTG1; CHP-212; CHP126; CHRF; CI-1; CMK; CMS; COLO-699; CTV-1; CTV-1: PP2; Cal-12T; Cal-148; Calu-3; CaoV3; Colo-704; Colo-824; Colo680N; DBTRG-05MG; DK-MG; DMS153; DMS 53; DMS 79; DND-41; DU-4475; DU.528; DU145; DV-90; Detroit562; EFM-19; EFM-192A; EFO-21; EFO-27; ELF-153; ENT01; ENT02; ENT03; ENT04; ENT05; ENT10; ENT12; ENT14; ENT15; ENT16; ENT17; ENT18; ENT19; ENT20; ENT23; ENT25; ENT26; ENT7; ENT8; ENT9; EOL-1; ES2; EVSA-T; FUOV1; GAMG; GI-CA-N; GMS-10; H128; H1299; H1355; H1373; H1417; H1435; H1437; H1563; H1648; H1650; H1666; H1693; H1703; H1734; H1781; H1793; H1838; H1869; H1915; H1944; H1975; H1993; H2023; H2030; H2052; H2066; H2085; H209; H2135; H2170; H2172; H2286; H2342; H2347; H2452; H28; H3255; H358; H4; H441; H446; H460; H520; H524; H526; H596; H647; H661; H810; H82; H838; H929; HCC1143; HCC1187; HCC1395; HCC1419; HCC1428; HCC15; HCC1500; HCC1569; HCC1599; HCC1806; HCC1937; HCC202; HCC366; HCC38; HCC44; HCC70; HCC78; HCC78: TSA 24h; HCC827; HCC827: Geldanamycin; HCC827: TSA; HCT116; HCT15; HCT15: TSA; HCT8; HCT8: TSA; HD-MyZ; HDLM-2; HDQ-P1; HEL; HEL: Flt3 inhibitor; HEL: Jak Inhibitor I; HL107A; HL107B; HL116A; HL116B; HL117A; HL117B; HL127A; HL127B; HL129A; HL130A; HL131A; HL131B; HL132A; HL132B; HL133A; HL137A; HL144A; HL144B; HL145A; HL145B; HL146A; HL146B; HL148A; HL148B; HL150A; HL150B; HL151A; HL151B; HL152B; HL183A; HL183B; HL184A; HL184B; HL213A; HL226A; HL226B; HL233A; HL233B; HL234A; HL234B; HL235A; HL25A; HL53A; HL53B; HL55A; HL55B; HL57; HL59A; HL59B; HL61A; HL61B; HL61b; HL66A; HL66B; HL68A; HL75A; HL76A; HL76B; HL79A; HL79B; HL83A; HL84A; HL84B; HL87A; HL87B; HL92A; HL92B; HL94A; HL94B; HL97A; HL97B; HL98A; HP28; HPAC: EGF; HT29; HU-3; Hs.683; Hs746T; Hs766T; IMR32; J82; JIMT-1; June07cs148; June07cs161; June07cs180; Jurkat: anti-CD3, anti-mouse Ig, anti-CD28; Jurkat: calyculin, pervanadate; Jurkat: pervanadate; Jurkat: pervanadate, calyculin; K562; KATO III; KBM-3; KELLY; KG-1; KMS-27; KOPT-K1; KPL-1; KY821; Karpas 299; Karpas-1106P; Kyse140; Kyse180; Kyse270; Kyse30; Kyse410; Kyse450; Kyse510; Kyse520; Kyse70; L428; L540; LAN-1; LAN-5; LCLC-103H; LN-405; LN18; LN229; LNCaP; LOU-NH91; LP-1; LXF-289; M-07e; M059J; M059K; MC-116; MCF7; MDA-MB-134vi; MDA-MB-157; MDA-MB-175vii; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; MDA-MB-468: EGF; MDAH2774; MEC-2; MHH-CALL4; MHH-NB-11; MIAPaCa-2; MIAPaCa-2: EGF; MKN-45; MKPL-1; ML-1; MNNG/MOS; MONO-MAC-6; MT-3; MUTZ-5; MV4-11; MV4-11: DMSO; MV4-11: SAHA 3h; MV4-11: SAHA 6h; MV4-11∥pervanadate; Marimo; Me-F2; Molm 14; Molt 15; N06211(1); N06218(1); N06218(2); N06BJ504(1); N06BJ504-R; N06BJ505(2); N06BJ526(21); N06BJ530(6); N06BJ573(9); N06BJ591(11); N06BJ593(13); N06BJ601(18); N06BJ606(19); N06C45AG-R; N06CSO2; N06CSO6; N06CS09; N06CS106; N06CS107; N06CS110AG-R; N06CS113-R; N06CS16; N06CS17; N06CS22(2)-R; N06CS22-1; N06CS22-2; N06CS23; N06CS34; N06CS38; N06CS39; N06CS40; N06CS75; N06CS77; N06CS82; N06CS83; N06CS87; N06CS89; N06CS90; N06CS91; N06CS93-1; N06CS93-2; N06CS94; N06CS97; N06CS97-R; N06CS98; N06CS98-2; N06N101; N06N 102 ; N06N 103 ; N06N 106 ; N06N109; N06N121; N06N127; N06N128; N06N129; N06N130; N06N131; N06N132; N06N75; N06N80; N06N90; N06N93; N06bj523(3); N06bj567(7); N06bj570(8); N06bj590(10); N06bj592(12); N06bj594(14); N06bj595(15); N06bj596(16); N06bj598(17); N06bj632(24); N06bj638(26); N06bj639(27); N06bj667(29); N06c144; N06c78; N06cs109; N06cs110; N06cs110-R; N06cs111; N06cs112; N06cs113; N06cs115; N06cs117; N06cs121; N06cs122; N06cs122-R; N06cs123; N06cs123(2); N06cs126; N06cs128; N06cs129; N06cs130; N06cs132; N06cs133; N06cs21; N06cs49; N06cs59; N06cs63; N06cs88; N06cs92; N06cs95; N87; N87: EGF; NALM-19; NCI-H716; NKM-1; Nomo-1; Nomo-1: DMSO; Nomo-1: SAHA 3h; Nomo-1: SAHA 6h; OCI-M1; OCI/AML2; OCI/AML3; OV90; PA-1; PL21; PL21∥pervanadate; Pfeiffer; RC-K8; RI-1; RKO: mutBRaf; RPMI-8266; RS4; SEM; SH-SY5Y; SIMA; SK-BR-3; SK-N-AS; SK-N-BE(2); SK-N-DZ; SK-N-FI; SK-N-MC; SK-N-SH; SK-OV-3; SNB-19; SNU-1; SNU-16; SNU-5; SNU-C2B; SNU-C2B: TSA; SU-DHL1; SU-DHL4; SUP-T13; SW1088; SW1710; SW1783; SW480; SW620; SW620: TSA; SW780; SW780; Scaber; SuDHL5; SuDHL8; T17; T98G; Thom; Thom(MPL, W515L); U118 MG; UACC-812; UACC-893; UM-UC-1; UT-7; VAC0432: mutBRaf; VAL; WSU-NHL; ZR-75-1; ZR-75-30; brain; brain: ischemia; cs001; cs012; cs015; cs018; cs019; cs024; cs025; cs026; cs029; cs037; cs041; cs042; cs048; cs057; cs068; cs069; cs070; cs103; cs104; cs105; cs106; cs107; cs110; cs111; cs114; cs131; cs133; cs136; cs153; csBC001; csC43; csC44; csC45; csC50; csC52; csC56; csC58; csC60; csC62; csC66; csC71; gz21; gz30; gz33; gz42; gz47; gz52; gz56; gz58; gz61; gz62; gz63; gz68; gz7; gz70; gz73; gz74; gz75; gzB1; h2073; h2228; lung (mouse); mouse heart; mouse liver; sw48; and sw48: TSA. In addition to the newly discovered phosphorylation sites (all having a phosphorylatable tyrosine), many known phosphorylation sites were also identified. 
     The immunoaffinity/mass spectrometric technique described in Rush et al, i.e., the “IAP” method, is described in detail in the Examples and briefly summarized below. 
     The IAP method 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, e.g., using SILAC or AQUA, may also be used to quantify isolated peptides in order to compare peptide levels in a sample to a baseline. 
     In the IAP method as disclosed herein, a general phosphotyrosine-specific monoclonal antibody (commercially available from Cell Signaling Technology, Inc., Beverly, Mass., Cat #9411 (p-Tyr-100)) may be used in the immunoaffinity step to isolate the widest possible number of phospho-tyrosine containing peptides from the cell extracts. 
     As described in more detail in the Examples, lysates may be prepared from various carcinoma cell lines or tissue samples and digested with trypsin after treatment with DTT and iodoacetamide to alkylate cysteine residues. Before the immunoaffinity step, peptides may be pre-fractionated (e.g., by reversed-phase solid phase extraction using Sep-Pak C 18  columns) to separate peptides from other cellular components. The solid phase extraction cartridges may then be eluted (e.g., with acetonitrile). Each lyophilized peptide fraction can be redissolved and treated with phosphotyrosine-specific antibody (e.g., P-Tyr-100, CST #9411) immobilized on protein Agarose. Immunoaffinity-purified peptides can be eluted and a portion of this fraction may be concentrated (e.g., with Stage or Zip tips) and analyzed by LC-MS/MS (e.g., using a ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer or LTQ). MS/MS spectra can be evaluated using, e.g., the program Sequest with the NCBI human protein database. 
     The novel phosphorylation sites identified are summarized in Table1/ FIG. 2 . Column A lists the parent (signaling) protein in which the phosphorylation site occurs. Column D identifies the tyrosine residue at which phosphorylation occurs (each number refers to the amino acid residue position of the tyrosine in the parent human protein, according to the published sequence retrieved by the SwissProt accession number). Column E shows flanking sequences of the identified tyrosine residues (which are the sequences of trypsin-digested peptides).  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. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Novel Phosphorylation Sites in Carcinoma. 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 A 
                 B 
                 C 
                 D 
                 E 
                 H 
                   
               
               
                   
                 Protein 
                 Accession 
                 Protein 
                 Phospho- 
                 Phosphorylation 
                 SEQ ID 
               
               
                   1 
                 Name 
                 No. 
                 Type 
                 Residue 
                 Site Sequence 
                 NO 
               
               
                   
               
               
                   2 
                 envoplakin 
                 NP_001979.1 
                 Adaptor/scaffold 
                 Y1352 
                 AAEDAVyELQSK 
                 SEQ ID NO: 1 
                   
               
               
                   
               
               
                   3 
                 IRS-4 
                 NP_003595.1 
                 Adaptor/scaffold 
                 Y291 
                 CGHSEQyFFLEVGR 
                 SEQ ID NO: 3 
               
               
                   
               
               
                   4 
                 OSTF1 
                 NP_036515.3 
                 Adaptor/scaffold 
                 Y207 
                 TLSNAEDyLDDEDSD 
                 SEQ ID NO: 4 
               
               
                   
               
               
                   5 
                 PHIP 
                 NP_060404.3 
                 Adaptor/scaffold 
                 Y505 
                 SyFNMIEGQGHGAVFDCK 
                 SEQ ID NO: 5 
               
               
                   
               
               
                   6 
                 RbBP1 
                 NP_002883.2 
                 Adaptor/scaffold 
                 Y20 
                 yRGAFCEAKIKTVK 
                 SEQ ID NO: 6 
               
               
                   
               
               
                   7 
                 SHANK3 
                 XP_037493.5 
                 Adaptor/scaffold 
                 Y127 
                 VyAQNLIDDKQFAK 
                 SEQ ID NO: 7 
               
               
                   
               
               
                   8 
                 VANGL1 
                 NP_620409.1 
                 Adaptor/scaffold 
                 Y312 
                 HMAGLKVyNVDGPSNNATGQSR 
                 SEQ ID NO: 9 
               
               
                   
               
               
                   9 
                 WAC 
                 NP_057712.2 
                 Adaptor/scaffold 
                 Y187 
                 MAVNSFPKDRDyR 
                 SEQ ID NO: 10 
               
               
                   
               
               
                  10 
                 CDH11 
                 NP_001788.2 
                 Adhesion or 
                 Y700 
                 DIKPEyQYMPR 
                 SEQ ID NO: 11 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  11 
                 CDH3 
                 NP_001784.2 
                 Adhesion or 
                 Y700 
                 DNVFyYGEEGGGEEDQDYDITQLHR 
                 SEQ ID NO: 12 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  12 
                 CLEC4F 
                 NP_775806.2 
                 Adhesion or 
                 Y482 
                 FNGGSLYyFSSVK 
                 SEQ ID NO: 13 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  13 
                 COL4A1 
                 NP_001836.1 
                 Adhesion or 
                 Y348 
                 GERGyPGTPGPR 
                 SEQ ID NO: 14 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  14 
                 CSPG2 
                 NP_004376.2 
                 Adhesion or 
                 Y208 
                 yPIRAPRVGCYGDK 
                 SEQ ID NO: 15 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  15 
                 CTGF 
                 NP_001892.1 
                 Adhesion or 
                 Y340 
                 TCACHYNCPGDNDIFESLyYR 
                 SEQ ID NO: 16 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  16 
                 DLL1 
                 NP_005609.2 
                 Adhesion or 
                 Y641 
                 YPAVDyNLVQDLK 
                 SEQ ID NO: 17 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  17 
                 DSC3 
                 NP_001932.1 
                 Adhesion or 
                 Y816 
                 yTYSEWHSFTQPR 
                 SEQ ID NO: 18 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  18 
                 DSC3 
                 NP_001932.1 
                 Adhesion or 
                 Y818 
                 GGHTEVDNCRYTySEWHSFTQPR 
                 SEQ ID NO: 19 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  19 
                 DSG1 
                 NP_001933.1 
                 Adhesion or 
                 Y672 
                 TSGMPEICQEySGTLR 
                 SEQ ID NO: 20 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  20 
                 ECM2 
                 NP_001384.1 
                 Adhesion or 
                 Y544 
                 YNKIEENRIAPLAWINQENLESIDLSyNK 
                 SEQ ID NO: 21 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  21 
                 Erbin 
                 NP_061165.1 
                 Adhesion or 
                 Y52 
                 TLEELyLDANQIEELPK 
                 SEQ ID NO: 22 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  22 
                 FBN1 
                 NP_000129.2 
                 Adhesion or 
                 Y2849 
                 yDKDYLSGELGDNLKMK 
                 SEQ ID NO: 23 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  23 
                 ITGB4 
                 NP_000204.3 
                 Adhesion or 
                 Y50 
                 DCAyCTDEMFRDR 
                 SEQ ID NO: 24 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  24 
                 ITGB8 
                 NP_002205.1 
                 Adhesion or 
                 Y447 
                 CDVTGGKNyAIIK 
                 SEQ ID NO: 25 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  25 
                 KIRREL 
                 NP_060710.2 
                 Adhesion or 
                 Y497 
                 GPASDyGPEPTPPGPAAPAGTDTTSQLSYE 
                 SEQ ID NO: 26 
               
               
                   
                   
                   
                 extracellular 
                   
                 NYEK 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  26 
                 KIRREL 
                 NP_060710.2 
                 Adhesion or 
                 Y574 
                 FSyTSQHSDYGQR 
                 SEQ ID NO: 27 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  27 
                 KIRREL 
                 NP_060710.2 
                 Adhesion or 
                 Y581 
                 FSYTSQHSDyGQR 
                 SEQ ID NO: 28 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  28 
                 LRRC7 
                 NP_065845.1 
                 Adhesion or 
                 Y52 
                 TLEELyLDANQIEELPK 
                 SEQ ID NO: 29 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  29 
                 MAG 
                 NP_002352.1 
                 Adhesion or 
                 Y571 
                 ISGAPEKyESERRLGSER 
                 SEQ ID NO: 30 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  30 
                 nectin 1 
                 NP_002846.3 
                 Adhesion or 
                 Y436 
                 AGPLGGSSyEEEEEEEEGGGGGERK 
                 SEQ ID NO: 31 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  31 
                 PCDH19 
                 NP_065817.1 
                 Adhesion or 
                 Y550 
                 DLEQyVNNVNNGPTRPSEAEPR 
                 SEQ ID NO: 32 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  32 
                 PCDH7 
                 NP_002580.2 
                 Adhesion or 
                 Y206 
                 AGAADSAPyPGGGGNGASGGGSGGSKRR 
                 SEQ ID NO: 33 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  33 
                 PCDHB12 
                 NP_061755.1 
                 Adhesion or 
                 Y247 
                 VVVVDINDNSPEFEQAFyEVK 
                 SEQ ID NO: 34 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  34 
                 Plakophilin 
                 NP_000290.2 
                 Adhesion or 
                 Y104 
                 FQAGNGSWGYPIyNGTLKR 
                 SEQ ID NO: 35 
               
               
                   
                 1 
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  35 
                 Plakophilin 
                 NP_000290.2 
                 Adhesion or 
                 Y499 
                 YRQLEyNAR 
                 SEQ ID NO: 36 
               
               
                   
                 1 
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  36 
                 Plakophilin 
                 NP_003619.2 
                 Adhesion or 
                 Y1062 
                 SEyDRTQPPMQYYNSOGDATHK 
                 SEQ ID NO: 37 
               
               
                   
                 4 
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  37 
                 ROBO1 
                 NP_002932.1 
                 Adhesion or 
                 Y1114 
                 QEVAPVQyNIVEQNK 
                 SEQ ID NO: 38 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  38 
                 SDK2 
                 NP_061937.3 
                 Adhesion or 
                 Y1762 
                 AHSFVNHYISDPTyYNSWR 
                 SEQ ID NO: 39 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  39 
                 TJP3 
                 NP_055243.1 
                 Adhesion or 
                 Y397 
                 ESSYDIyRVPSSQSMEDR 
                 SEQ ID NO: 40 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                  40 
                 PITPNM2 
                 NP_065896.1 
                 Receptor, 
                 Y191 
                 QVFPIMCAyK 
                 SEQ ID NO: 41 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  41 
                 cyclin B1 
                 NP_114172.1 
                 Cell cycle 
                 Y177 
                 DIYAyLRQLEEEQAVRPK 
                 SEQ ID NO: 42 
               
               
                   
                   
                   
                 regulation 
               
               
                   
               
               
                  42 
                 MLF1 
                 NP_071888.1 
                 Cell cycle 
                 Y206 
                 TGDEEVNQEFINMNESDAHAFDEEWQSEV 
                 SEQ ID NO: 43 
               
               
                   
                   
                   
                 regulation 
                   
                 LKyK 
               
               
                   
               
               
                  43 
                 NDN 
                 NP_002478.1 
                 Transcriptional 
                 Y132 
                 MIIWFPDMVKDVIGSyKK 
                 SEQ ID NO: 44 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                  44 
                 APCS 
                 NP_001630.1 
                 Chaperone 
                 Y142 
                 GLRQGyFVEAQPK 
                 SEQ ID NO: 45 
               
               
                   
               
               
                  45 
                 HSC70 
                 NP_006588.1 
                 Chaperone 
                 Y41 
                 TTPSyVAFTDTER 
                 SEQ ID NO: 46 
               
               
                   
               
               
                  46 
                 HSPA4L 
                 NP_055093.2 
                 Chaperone 
                 Y663 
                 LEDTENWLyEDGEDQPK 
                 SEQ ID NO: 48 
               
               
                   
               
               
                  47 
                 hnRNP D- 
                 NP_005454.1 
                 Chromatin, DNA- 
                 Y314 
                 CEIKVAQPKEVyR 
                 SEQ ID NO: 50 
               
               
                   
                 like 
                   
                 binding, DNA 
               
               
                   
                   
                   
                 repair or DNA 
               
               
                   
                   
                   
                 replication 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  48 
                 MSH4 
                 NP_002431.2 
                 Chromatin, DNA- 
                 Y132 
                 DTNYPQTLKTPLSTGNPQRSGyK 
                 SEQ ID NO: 51 
               
               
                   
                   
                   
                 binding, DNA 
               
               
                   
                   
                   
                 repair or DNA 
               
               
                   
                   
                   
                 replication 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  49 
                 NAP1L1 
                 NP_004528.1 
                 Chromatin, DNA- 
                 Y106 
                 KyAVLYQPLFDK 
                 SEQ ID NO: 52 
               
               
                   
                   
                   
                 binding, DNA 
               
               
                   
                   
                   
                 repair or DNA 
               
               
                   
                   
                   
                 replication 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  50 
                 NPAS3 
                 NP_071406.1 
                 Transcriptional 
                 Y406 
                 NIIWVNYLLSNPEyK 
                 SEQ ID NO: 53 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                  51 
                 actin, alpha 
                 NP_001091.1 
                 Cytoskeletal 
                 Y71 
                 GILTLKyPIEHGIITNWDDMEK 
                 SEQ ID NO: 54 
               
               
                   
                 1 
                   
                 protein 
               
               
                   
               
               
                  52 
                 actin, beta 
                 NP_001092.1 
                 Cytoskeletal 
                 Y53 
                 HQGVMVGMGQKDSyVGDEAQSKR 
                 SEQ ID NO: 55 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  53 
                 AIF1 
                 NP_001614.3 
                 Cytoskeletal 
                 Y124 
                 MILMyEEKAREK 
                 SEQ ID NO: 56 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  54 
                 CK17 
                 NP_000413.1 
                 Cytoskeletal 
                 Y98 
                 LASyLDKVR 
                 SEQ ID NO: 57 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  55 
                 CK6 
                 NP_375109.1 
                 Cytoskeletal 
                 Y356 
                 SRAEAESWyQTKYEELQVTAGR 
                 SEQ ID NO: 58 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  56 
                 CK6 
                 NP_775109.1 
                 Cytoskeletal 
                 Y360 
                 SRAEAESWYQTKyEELQVTAGR 
                 SEQ ID NO: 59 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  57 
                 CK6 
                 NP_775109.1 
                 Cytoskeletal 
                 Y551 
                 AIGGGLSSVGGGSSTIKyTTTSSSSR 
                 SEQ ID NO: 60 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  58 
                 cofilin 2 
                 NP_068733.1 
                 Cytoskeletal 
                 Y117 
                 MIyASSKDAIKK 
                 SEQ ID NO: 62 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  59 
                 cofilin 2 
                 NP_068733.1 
                 Cytoskeletal 
                 Y85 
                 YALyDATYETK 
                 SEQ ID NO: 63 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  60 
                 cofilin 2 
                 NP_068733.1 
                 Cytoskeletal 
                 Y89 
                 YALYDATyETKESK 
                 SEQ ID NO: 64 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  61 
                 desmoplakin 
                 NP_002221.1 
                 Cytoskeletal 
                 Y644 
                 NEGTATyAAAVLFR 
                 SEQ ID NO: 65 
               
               
                   
                 3 
                   
                 protein 
               
               
                   
               
               
                  62 
                 K6IRS3 
                 NP_778238.1 
                 Cytoskeletal 
                 Y325 
                 SKAEAEALyQTK 
                 SEQ ID NO: 67 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  63 
                 KRT14 
                 NP_000517.2 
                 Cytoskeletal 
                 Y129 
                 LASyLDKVR 
                 SEQ ID NO: 68 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  64 
                 KRT15 
                 NP_002266.2 
                 Cytoskeletal 
                 Y119 
                 LASyLDKVR 
                 SEQ ID NO: 69 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  65 
                 KRT16 
                 NP_005548.2 
                 Cytoskeletal 
                 Y131 
                 LASyLDKVR 
                 SEQ ID NO: 70 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  66 
                 KRT23 
                 NP_056330.3 
                 Cytoskeletal 
                 Y354 
                 QNNEyQVLLGIK 
                 SEQ ID NO: 71 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  67 
                 lamin B2 
                 NP_116126.2 
                 Cytoskeletal 
                 Y299 
                 LESLSyQLSGLQK 
                 SEQ ID NO: 72 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  68 
                 MAP1B 
                 NP_005900.1 
                 Cytoskeletal 
                 Y1174 
                 YESSLYSQEySKPADVTPLNGFSEGSK 
                 SEQ ID NO: 73 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  69 
                 moesin 
                 NP_002435.1 
                 Cytoskeletal 
                 Y270 
                 APDFVFyAPR 
                 SEQ ID NO: 75 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  70 
                 peripherin 
                 NP_006253.2 
                 Cytoskeletal 
                 Y287 
                 NLQEAEEWyK 
                 SEQ ID NO: 76 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  71 
                 plectin 1 
                 NP_000436.2 
                 Cytoskeletal 
                 Y1316 
                 QYINAIKDYELQLVTyK 
                 SEQ ID NO: 77 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  72 
                 radixin 
                 NP_002897.1 
                 Cytoskeletal 
                 Y270 
                 APDFVFyAPR 
                 SEQ ID NO: 78 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  73 
                 RAI14 
                 NP_056392.1 
                 Cytoskeletal 
                 Y519 
                 LGLVSPESMDNySHFHELR 
                 SEQ ID NO: 79 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  74 
                 RAI14 
                 NP_056392.1 
                 Cytoskeletal 
                 Y583 
                 SSyCSVIENMNK 
                 SEQ ID NO: 80 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  75 
                 supervillin 
                 NP_0031651 
                 Cytoskeletal 
                 Y120 
                 QLAEKyGLTLDPEADSEYLSR 
                 SEQ ID NO: 81 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  76 
                 SYNE1 
                 NP_149062.1 
                 Cytoskeletal 
                 Y3888 
                 GEALLELVQDVTLKDKIDQLQSDyQDLCSIG 
                 SEQ ID NO: 82 
               
               
                   
                   
                   
                 protein 
                   
                 K 
               
               
                   
               
               
                  77 
                 TMOD3 
                 NP_055362.1 
                 Cytoskeletal 
                 Y12 
                 DLEKyKDLDEDELLGNLSETELK 
                 SEQ ID NO: 83 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                  78 
                 ACADSB 
                 NP_001600.1 
                 Enzyme, misc. 
                 Y199 
                 ADKEGDYyVLNGSK 
                 SEQ ID NO: 84 
               
               
                   
               
               
                  79 
                 ACSL5 
                 NP_057318.2 
                 Enzyme, misc. 
                 Y608 
                 KNIFKLAQGEyIAPEK 
                 SEQ ID NO: 85 
               
               
                   
               
               
                  80 
                 AKR1C1 
                 NP_001344.2 
                 Enzyme, misc. 
                 Y55 
                 HIDSAHLyNNEEQVGLAIR 
                 SEQ ID NO: 86 
               
               
                   
               
               
                  81 
                 AKR1C2 
                 NP_001345.1 
                 Enzyme, misc. 
                 Y81 
                 REDIFyTSK 
                 SEQ ID NO: 87 
               
               
                   
               
               
                  82 
                 AKR1C3 
                 NP_003730.4 
                 Enzyme, misc. 
                 Y81 
                 REDIFyTSK 
                 SEQ ID NO: 88 
               
               
                   
               
               
                  83 
                 AKR1C4 
                 NP_001809.2 
                 Enzyme, misc. 
                 Y24 
                 LNDGHFMPVLGFGTyAPPEVPR 
                 SEQ ID NO: 89 
               
               
                   
               
               
                  84 
                 AKR1C4 
                 NP_001809.2 
                 Enzyme, misc. 
                 Y81 
                 REDIFyTSK 
                 SEQ ID NO: 90 
               
               
                   
               
               
                  85 
                 ATE1 
                 NP_001001976.1 
                 Enzyme, misc. 
                 Y433 
                 GQYRPSDLLCPETyVWVPIEQCLPSLENSK 
                 SEQ ID NO: 91 
               
               
                   
               
               
                  86 
                 CYP24A1 
                 NP_000773.2 
                 Enzyme, misc. 
                 Y220 
                 WSFESICLVLyEK 
                 SEQ ID NO: 92 
               
               
                   
               
               
                  87 
                 EPRS 
                 NP_004437.2 
                 Enzyme, misc. 
                 Y423 
                 KPYIWEySR 
                 SEQ ID NO: 93 
               
               
                   
               
               
                  88 
                 GGPS1 
                 NP_004828.1 
                 Enzyme, misc. 
                 Y272 
                 ELEAKAyKQIDAR 
                 SEQ ID NO: 94 
               
               
                   
               
               
                  89 
                 GSTA2 
                 NP_000837.2 
                 Enzyme, misc. 
                 Y49 
                 SAEDLDKLRNDGyLMFQQVPMVEIDGMK 
                 SEQ ID NO: 95 
               
               
                   
               
               
                  90 
                 LIPF 
                 NP_004181.1 
                 Enzyme, misc. 
                 Y196 
                 TFyALAPVATVK 
                 SEQ ID NO: 97 
               
               
                   
               
               
                  91 
                 LTB4DH 
                 NP_036344.1 
                 Enzyme, misc. 
                 Y16 
                 HFVGyPTNSDFELK 
                 SEQ ID NO: 98 
               
               
                   
               
               
                  92 
                 NNT 
                 NP_036475.2 
                 Enzyme, misc. 
                 Y373 
                 GITHIGyTDLPSR 
                 SEQ ID NO: 99 
               
               
                   
               
               
                  93 
                 OGT 
                 NP_858059.1 
                 Enzyme, misc. 
                 Y966 
                 VAASQLTCLGCLELIAKNRQEyEDIAVK 
                 SEQ ID NO: 100 
               
               
                   
               
               
                  94 
                 PDHA2 
                 NP_005381.1 
                 Enzyme, misc. 
                 Y299 
                 YHGHSMSDPGVSyRTREEIQEVR 
                 SEQ ID NO: 101 
               
               
                   
               
               
                  95 
                 PGD 
                 NP_002622.2 
                 Enzyme, misc. 
                 Y481 
                 PGQFIHTNWTGHGGTVSSSSyNA 
                 SEQ ID NO: 102 
               
               
                   
               
               
                  96 
                 PLA2G4B 
                 NP_005081.1 
                 Enzyme, misc. 
                 Y31 
                 ELCVPLAVPyLDK 
                 SEQ ID NO: 103 
               
               
                   
               
               
                  97 
                 RDH10 
                 NP_742034.1 
                 Enzyme, misc. 
                 Y322 
                 FLGADKCMyPFIAQR 
                 SEQ ID NO: 104 
               
               
                   
               
               
                  98 
                 UBE2J1 
                 NP_057105.2 
                 Ubiquitin 
                 Y312 
                 IYLANEyIFDFEL 
                 SEQ ID NO: 105 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                  99 
                 DOCK10 
                 GI: 32469767 
                 G protein or 
                 Y2073 
                 LQGSVSVKVNAGPMAyAR 
                 SEQ ID NO: 106 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 100 
                 RAB6IP1 
                 NP_056028.2 
                 G protein or 
                 Y719 
                 EKyIQEAR 
                 SEQ ID NO: 107 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 101 
                 RASAL2 
                 NP_004832.1 
                 G protein or 
                 Y672 
                 SPSQDNTDSyFR 
                 SEQ ID NO: 108 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 102 
                 RGS17 
                 NP_036551.3 
                 G protein or 
                 Y171 
                 NLLDPNPHMyEDAQLQIYTLMHR 
                 SEQ ID NO: 109 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 103 
                 SOS2 
                 NP_008870.1 
                 G protein or 
                 Y679 
                 KEyVQPVQLRVLNVFR 
                 SEQ ID NO: 110 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 104 
                 SRGAP1 
                 NP_065813.1 
                 G protein or 
                 Y359 
                 yQQLQSRLATLKIENEEVK 
                 SEQ ID NO: 111 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 105 
                 SPP2 
                 NP_008875.1 
                 Inhibitor protein 
                 Y109 
                 DyYVSTAVCR 
                 SEQ ID NO: 112 
               
               
                   
               
               
                 106 
                 B-CK 
                 NP_001814.2 
                 Kinase (non- 
                 Y269 
                 SKDyEFMWNPHLGYILTCPSNLGTGLR 
                 SEQ ID NO: 113 
               
               
                   
                   
                   
                 protein) 
               
               
                   
               
               
                 107 
                 GK2 
                 NP_149991.2 
                 Kinase (non- 
                 Y500 
                 FEPQIQATESEIRyATWKK 
                 SEQ ID NO: 114 
               
               
                   
                   
                   
                 protein) 
               
               
                   
               
               
                 108 
                 PI4KII 
                 NP_060895.1 
                 Kinase (non- 
                 Y138 
                 IYQGSSGSyFVKDPQGRIIAVFK 
                 SEQ ID NO: 115 
               
               
                   
                   
                   
                 protein) 
               
               
                   
               
               
                 109 
                 PIK3R2 
                 NP_005018.1 
                 Kinase (non- 
                 Y74 
                 GDFPGTyVEFLGPVALAR 
                 SEQ ID NO: 116 
               
               
                   
                   
                   
                 protein) 
               
               
                   
               
               
                 110 
                 PIK4CA 
                 NP_002641.1 
                 Kinase (non- 
                 Y842 
                 SRTyDMIQYYQNDIPY 
                 SEQ ID NO: 117 
               
               
                   
                   
                   
                 protein) 
               
               
                   
               
               
                 111 
                 ephrin-B1 
                 NP_004420.1 
                 Ligand, receptor 
                 Y344 
                 VSGDYGHPVYIVQEMPPQSPANIYyKV 
                 SEQ ID NO: 118 
               
               
                   
                   
                   
                 tyrosine kinase 
               
               
                   
               
               
                 112 
                 ephrin-B2 
                 NP_004084.1 
                 Ligand, receptor 
                 Y11 
                 yCWGVLMVLCR 
                 SEQ ID NO: 119 
               
               
                   
                   
                   
                 tyrosine kinase 
               
               
                   
               
               
                 113 
                 NRG1 
                 NP_039250.1 
                 Ligand, receptor 
                 Y530 
                 IVEDEEyETTQEYEPAQEPVKK 
                 SEQ ID NO: 120 
               
               
                   
                   
                   
                 tyrosine kinase 
               
               
                   
               
               
                 114 
                 SDPR 
                 NP_004648.1 
                 Lipid binding 
                 Y399 
                 VRYEGSyALTSEEAERSDGDPVQPAVLQVH 
                 SEQ ID NO: 121 
               
               
                   
                   
                   
                 protein 
                   
                 QTS 
               
               
                   
               
               
                 115 
                 CHCHD3 
                 NP_060282.1 
                 Mitochondrial 
                 Y53 
                 YSGAyGASVSDEELK 
                 SEQ ID NO: 122 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 116 
                 CLPX 
                 NP_006651.2 
                 Mitochondrial 
                 Y529 
                 NAVIPQyQALFSMDK 
                 SEQ ID NO: 123 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 117 
                 CS 
                 NP_004068.2 
                 Mitochondrial 
                 Y80 
                 GMKGLVyETSVLDPDEGIR 
                 SEQ ID NO: 124 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 118 
                 ETFB 
                 NP_001976.1 
                 Mitochondrial 
                 Y192 
                 yATLPNIMKAK 
                 SEQ ID NO: 125 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 119 
                 HMGCL 
                 NP_000182.2 
                 Mitochondrial 
                 Y198 
                 KFYSMGCyEISLGDTIGVGTPGIMK 
                 SEQ ID NO: 126 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 120 
                 MRPL9 
                 NP_113608.1 
                 Mitochondrial 
                 Y80 
                 RHRVyKLVEDTK 
                 SEQ ID NO: 127 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 121 
                 UCP1 
                 NP_068605.1 
                 Mitochondrial 
                 Y55 
                 LQVQGECPTSSVIRyK 
                 SEQ ID NO: 128 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 122 
                 CENPE 
                 NP_001804.2 
                 Motor or 
                 Y283 
                 LSDGQVGGFINyRDSKLTR 
                 SEQ ID NO: 129 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 123 
                 DNAH5 
                 NP_001360.1 
                 Motor or 
                 Y4244 
                 yMIGEIQYGGRVTDDYDKRLLNTFAK 
                 SEQ ID NO: 130 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 124 
                 KIF5B 
                 NP_004512.1 
                 Motor or 
                 Y649 
                 SLTEyLQNVEQK 
                 SEQ ID NO: 131 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 125 
                 MYH2 
                 NP_060004.2 
                 Motor or 
                 Y1381 
                 TKyETDAIQR 
                 SEQ ID NO: 132 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 126 
                 MYH2 
                 NP_060004.2 
                 Motor or 
                 Y1494 
                 NAyEESLDQLETLKR 
                 SEQ ID NO: 133 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 127 
                 MYH2 
                 NP_060004.2 
                 Motor or 
                 Y721 
                 GFPSRILyADFKQRYK 
                 SEQ ID NO: 134 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 128 
                 MYH4 
                 NP_060003.1 
                 Motor or 
                 Y1379 
                 TKyETDAIQR 
                 SEQ ID NO: 135 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 129 
                 MYH4 
                 NP_060003.1 
                 Motor or 
                 Y719 
                 GFPSRILyADFKQRYK 
                 SEQ ID NO: 136 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 130 
                 MYH8 
                 NP_002463.1 
                 Motor or 
                 Y1378 
                 TKyETDAIQR 
                 SEQ ID NO: 137 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 131 
                 MYH8 
                 NP_002463.1 
                 Motor or 
                 Y413 
                 VKVGNEyVTK 
                 SEQ ID NO: 138 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 132 
                 MYO10 
                 NP_036466.1 
                 Motor or 
                 Y1248 
                 LMyFENDSEEKLK 
                 SEQ ID NO: 139 
               
               
                   
                   
                   
                 contractile 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 133 
                 MTM1 
                 NP_000243.1 
                 Phosphatase 
                 Y260 
                 yLDVIRETNK 
                 SEQ ID NO: 140 
               
               
                   
               
               
                 134 
                 PPM1D 
                 NP_003611.1 
                 Phosphatase 
                 Y308 
                 HKyIILGSDGLWNMIPPQDAISMCQDQEEKK 
                 SEQ ID NO: 141 
               
               
                   
               
               
                 135 
                 TIMM50 
                 NP_001001563.1 
                 Phosphatase 
                 Y165 
                 RAPDQAAEIGSRGSTKAQGPQQQPGSEGP 
                 SEQ ID NO: 142 
               
               
                   
                   
                   
                   
                   
                 SyAK 
               
               
                   
               
               
                 136 
                 ACE2 
                 NP_068576.1 
                 Protease 
                 Y781 
                 SGENPyASIDISK 
                 SEQ ID NO: 143 
               
               
                   
               
               
                 137 
                 BACE 
                 NP_036236.1 
                 Protease 
                 Y260 
                 REWYyEVIIVR 
                 SEQ ID NO: 144 
               
               
                   
               
               
                 138 
                 CFI 
                 NP_000195.1 
                 Protease 
                 Y22 
                 FCKVTyTSQEDLVEK 
                 SEQ ID NO: 145 
               
               
                   
               
               
                 139 
                 CTSB 
                 NP_001899.1 
                 Protease 
                 Y33 
                 SRPSFHPLSDELVNyVNK 
                 SEQ ID NO: 146 
               
               
                   
               
               
                 140 
                 MMP16 
                 NP_005932.2 
                 Protease 
                 Y521 
                 FNNQILKVEPGyPR 
                 SEQ ID NO: 147 
               
               
                   
               
               
                 141 
                 PSMD12 
                 NP_002807.1 
                 Protease 
                 Y20 
                 MEVDySATVDQR 
                 SEQ ID NO: 148 
               
               
                   
               
               
                 142 
                 PSMD5 
                 NP_005038.1 
                 Protease 
                 Y478 
                 TIAEIFGNPNyLR 
                 SEQ ID NO: 149 
               
               
                   
               
               
                 143 
                 TPSB2 
                 NP_077078.5 
                 Protease 
                 Y97 
                 EQHLYyQDQLLPVSR 
                 SEQ ID NO: 150 
               
               
                   
               
               
                 144 
                 TPSD1 
                 NP_036349.1 
                 Protease 
                 Y104 
                 EQHLYyQDQLLPVSR 
                 SEQ ID NO: 151 
               
               
                   
               
               
                 145 
                 ChaK2 
                 NP_060132.3 
                 Protein kinase, 
                 Y832 
                 KVyEFYSAPIVK 
                 SEQ ID NO: 152 
               
               
                   
                   
                   
                 atypical 
               
               
                   
               
               
                 146 
                 PDHK1 
                 NP_002601.1 
                 Protein kinase, 
                 Y244 
                 RLCDLYyINSPELELEELNAK 
                 SEQ ID NO: 153 
               
               
                   
                   
                   
                 atypical 
               
               
                   
               
               
                 147 
                 DYRK4 
                 NP_003836.1 
                 Protein kinase, 
                 Y262 
                 VyTYIQSR 
                 SEQ ID NO: 154 
               
               
                   
                   
                   
                 dual-specificity 
               
               
                   
               
               
                 148 
                 LRIG1 
                 NP_056356.2 
                 Protein kinase, 
                 Y1054 
                 AEAQyLLVSNGHLPK 
                 SEQ ID NO: 155 
               
               
                   
                   
                   
                 regulatory 
               
               
                   
                   
                   
                 subunit 
               
               
                   
               
               
                 149 
                 LRIG1 
                 NP_056356.2 
                 Protein kinase, 
                 Y941 
                 VVCSDCNTEVDCySR 
                 SEQ ID NO: 156 
               
               
                   
                   
                   
                 regulatory 
               
               
                   
                   
                   
                 subunit 
               
               
                   
               
               
                 150 
                 ASK1 
                 NP_005914.1 
                 Protein kinase, 
                 Y814 
                 DIKGDNVLINTySGVLKISDFGTSK 
                 SEQ ID NO: 157 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 151 
                 CdkL5 
                 NP_003150.1 
                 Protein kinase, 
                 Y482 
                 QSRHSYIDTIPQSSRSPSyR 
                 SEQ ID NO: 158 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 152 
                 CK2- 
                 NP_001887.1 
                 Protein kinase, 
                 Y240 
                 REPFFHGQDNyDQLVR 
                 SEQ ID NO: 159 
               
               
                   
                 alpha2 
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 153 
                 LRRK2 
                 NP_940980.2 
                 Protein kinase, 
                 Y636 
                 ILVSSLyRFK 
                 SEQ ID NO: 160 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 154 
                 MINK 
                 NP_056531.1 
                 Protein kinase, 
                 Y950 
                 LDQLQyDVR 
                 SEQ ID NO: 161 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 155 
                 PKR 
                 NP_002750.1 
                 Protein kinase, 
                 Y142 
                 MGQKEySIGTGSTK 
                 SEQ ID NO: 162 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 156 
                 Titin 
                 NP_596869.2 
                 Protein kinase, 
                 Y8598 
                 MyGITDFRGLLQAFELLK 
                 SEQ ID NO: 163 
               
               
                   
                   
                   
                 Ser/Thr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 157 
                 BMPR2 
                 NP_001195.2 
                 Protein kinase, 
                 Y708 
                 QFSGPDPLSSTSSSLLyPLIK 
                 SEQ ID NO: 164 
               
               
                   
                   
                   
                 Ser/Thr 
               
               
                   
                   
                   
                 (receptor) 
               
               
                   
               
               
                 158 
                 Fer 
                 NP_005237.1 
                 Protein kinase, 
                 Y200 
                 GAQLHQNQYyDITLPLLLDSLQK 
                 SEQ ID NO: 165 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 159 
                 Fes 
                 NP_001996.1 
                 Protein kinase, 
                 Y156 
                 KyQEASKDKDRDK 
                 SEQ ID NO: 166 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 160 
                 Fgr 
                 NP_005239.1 
                 Protein kinase, 
                 Y180 
                 GAySLSIR 
                 SEQ ID NO: 167 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 161 
                 Fyn 
                 NP_002028.1 
                 Protein kinase, 
                 Y185 
                 ESETTKGAySLSIR 
                 SEQ ID NO: 168 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 162 
                 Src 
                 NP_005408.1 
                 Protein kinase, 
                 Y439 
                 QGAKFPIKWTAPEAALyGR 
                 SEQ ID NO: 170 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 163 
                 Yes 
                 NP_005424.1 
                 Protein kinase, 
                 Y159 
                 NGYIPSNYVAPADSIQAEEWyFGK 
                 SEQ ID NO: 171 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 164 
                 Yes 
                 NP_005424.1 
                 Protein kinase, 
                 Y222 
                 KLDNGGyYITTR 
                 SEQ ID NO: 172 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 165 
                 Yes 
                 NP_005424.1 
                 Protein kinase, 
                 Y223 
                 KLDNGGYyITTR 
                 SEQ ID NO: 173 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 166 
                 Yes 
                 NP_005424.1 
                 Protein kinase, 
                 Y446 
                 QGAKFPIKWTAPEAALyGR 
                 SEQ ID NO: 174 
               
               
                   
                   
                   
                 Tyr (non- 
               
               
                   
                   
                   
                 receptor) 
               
               
                   
               
               
                 167 
                 EphA5 
                 NP_004430.3 
                 Protein kinase, 
                 Y790 
                 yLSDMGYVHRDLAAR 
                 SEQ ID NO: 175 
               
               
                   
                   
                   
                 Tyr (receptor) 
               
               
                   
               
               
                 168 
                 EphA5 
                 NP_004430.3 
                 Protein kinase, 
                 Y833 
                 VLEDDPEAAyTTR 
                 SEQ ID NO: 176 
               
               
                   
                   
                   
                 Tyr (receptor) 
               
               
                   
               
               
                 169 
                 EphA6 
                 XP_496653.1 
                 Protein kinase, 
                 Y274 
                 yLSDMGYVHRDLAAR 
                 SEQ ID NO: 177 
               
               
                   
                   
                   
                 Tyr (receptor) 
               
               
                   
               
               
                 170 
                 EphB1 
                 NP_004432.1 
                 Protein kinase, 
                 Y634 
                 VIGAGEFGEVyK 
                 SEQ ID NO: 178 
               
               
                   
                   
                   
                 Tyr (receptor) 
               
               
                   
               
               
                 171 
                 HER4 
                 NP_005226.1 
                 Protein kinase, 
                 Y1128 
                 PVAPHVQEDSSTQRySADPTVFAPERSPR 
                 SEQ ID NO: 179 
               
               
                   
                   
                   
                 Tyr (receptor) 
               
               
                   
               
               
                 172 
                 AQP1 
                 NP_932766.1 
                 Receptor, 
                 Y253 
                 VWTSGQVEEyDLDADDINSRVEMKPK 
                 SEQ ID NO: 180 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 173 
                 ATP2A1 
                 NP_004311.1 
                 Receptor, 
                 Y497 
                 SMSVyCSPAK 
                 SEQ ID NO: 181 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 174 
                 ATP2A1 
                 NP_775293.1 
                 Receptor, 
                 Y527 
                 GAPEGVIDRCNyVR 
                 SEQ ID NO: 182 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 175 
                 ATP6V0A1 
                 NP_005168.2 
                 Receptor, 
                 Y364 
                 MQTNQTPPTyNKTNK 
                 SEQ ID NO: 183 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 176 
                 BAI2 
                 NP_001694.1 
                 Receptor, 
                 Y267 
                 yGEEPEEEPKVK 
                 SEQ ID NO: 184 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 177 
                 CD229 
                 NP_002339.2 
                 Receptor, 
                 Y309 
                 EEAATADPLIKSRDPyK 
                 SEQ ID NO: 185 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 178 
                 CLCN7 
                 NP_001278.1 
                 Receptor, 
                 Y99 
                 YESLDyDNSENQLFLEEER 
                 SEQ ID NO: 186 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 179 
                 CNGB1 
                 NP_001288.1 
                 Receptor, 
                 Y897 
                 SCMDSTVKyMNFYKIPK 
                 SEQ ID NO: 187 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 180 
                 Cx43 
                 NP_000156.1 
                 Receptor, 
                 Y267 
                 YAyFNGCSSPTAPLSPMSPPGYK 
                 SEQ ID NO: 188 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 181 
                 Cx43 
                 NP_000156.1 
                 Receptor, 
                 Y286 
                 YAYFNGCSSPTAPLSPMSPPGyK 
                 SEQ ID NO: 189 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 182 
                 FAT2 
                 NP_001438.1 
                 Receptor, 
                 Y4223 
                 HSTPVVMPEPNGLyGGFPFPLEMENK 
                 SEQ ID NO: 190 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 183 
                 FAT2 
                 NP001438.1 
                 Receptor, 
                 Y4309 
                 AGPSyAVCEVEGAPLAGQGQPR 
                 SEQ ID NO: 191 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 184 
                 GABRB2 
                 NP_000804.1 
                 Receptor, 
                 Y98 
                 DKRLSyNVIPLNLTLDNR 
                 SEQ ID NO: 192 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 185 
                 GPC5 
                 NP_004457.1 
                 Receptor, 
                 Y257 
                 ALLKMQyCPHCQGLALTKPCMGYCLNVMR 
                 SEQ ID NO: 193 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 186 
                 GPR175 
                 NP_057456.1 
                 Receptor, 
                 Y312 
                 CQVDETEEPDVHLPQPyAVAR 
                 SEQ ID NO: 194 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 187 
                 HBB 
                 NP_000509.1 
                 Receptor, 
                 Y131 
                 EFTPPVQAAyQK 
                 SEQ ID NO: 195 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 188 
                 IL1RAP 
                 NP_002173.1 
                 Receptor, 
                 Y564 
                 SSSDEQGLSySSLK 
                 SEQ ID NO: 196 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 189 
                 KIR2DL2 
                 NP_055033.1 
                 Receptor, 
                 Y302 
                 TANSEDSDEQDPQEVTyTQLNHCVFTQR 
                 SEQ ID NO: 197 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 190 
                 latrophilin 1 
                 NP_055736.2 
                 Receptor, 
                 Y1380 
                 DSLyASGANLR 
                 SEQ ID NO: 198 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 191 
                 LRP5 
                 NP_002326.1 
                 Receptor, 
                 Y1552 
                 GMAPPTTPCSTDVCDSDySASR 
                 SEQ ID NO: 199 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 192 
                 myoferlin 
                 NP_038479.1 
                 Receptor, 
                 Y953 
                 YPGGDWKPAEDTyTDANGDK 
                 SEQ ID NO: 200 
               
               
                   
                   
                   
                 channel, or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 193 
                 NETO2 
                 NP_060562.3 
                 Receptor, 
                 Y422 
                 DKEISADLADLSEELDNyQK 
                 SEQ ID NO: 201 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 194 
                 NKCC2 
                 NP_000329.1 
                 Receptor, 
                 Y944 
                 LRIyVGGKINRIEEEK 
                 SEQ ID NO: 202 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 195 
                 Nup153 
                 NP_005115.2 
                 Receptor, 
                 Y301 
                 QLSAQSyGVTSSTARR 
                 SEQ ID NO: 203 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 196 
                 PGRMC1 
                 NP_006658.1 
                 Receptor, 
                 Y113 
                 KFYGPEGPyGVFAGR 
                 SEQ ID NO: 204 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 197 
                 PGRMC1 
                 NP_006658.1 
                 Receptor, 
                 Y180 
                 EGEEPTVySDEEEPKDESAR 
                 SEQ ID NO: 205 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 198 
                 PKHD1 
                 NP_619639.2 
                 Receptor, 
                 Y828 
                 yLNASDFTVK 
                 SEQ ID NO: 206 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 199 
                 PLVAP 
                 NP_112600.1 
                 Receptor, 
                 Y71 
                 RAEGLySQLLGLTASQSNLTKELNFTTR 
                 SEQ ID NO: 207 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 200 
                 SCN7A 
                 NP_002967.1 
                 Receptor, 
                 Y1532 
                 FDPDRTQyIDSSK 
                 SEQ ID NO: 208 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 201 
                 SLC1A3 
                 NP_004163.2 
                 Receptor, 
                 Y523 
                 KPyQLIAQDNETEKPIDSETKM 
                 SEQ ID NO: 209 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 202 
                 SLC25A4 
                 NP_001142.2 
                 Receptor, 
               
               
                   
                   
                   
                 channel, 
                 Y191 
                 AAyFGVYDTAK 
                 SEQ ID NO: 211 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 203 
                 SLC41A2 
                 NP_115524.2 
                 Receptor, 
                 Y29 
                 YDDyANYNYCDGR 
                 SEQ ID NO: 213 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 204 
                 SLC4A2 
                 NP_003031.2 
                 Receptor, 
                 Y73 
                 SYGEEDFEyHR 
                 SEQ ID NO: 214 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 205 
                 SLC7A8 
                 NP_036376.2 
                 Receptor, 
                 Y519 
                 GSGTEEANEDMEEQQQPMyQPTPTKDKDV 
                 SEQ ID NO: 215 
               
               
                   
                   
                   
                 channel, 
                   
                 AGQPQP 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 206 
                 SLC9A9 
                 NP_375924.1 
                 Receptor, 
                 Y622 
                 ASPQTPGKENIyEGDLGLGGYELK 
                 SEQ ID NO: 216 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 207 
                 SLITRK3 
                 NP_055741.2 
                 Receptor, 
                 Y966 
                 LQTKPDyLEVLEK 
                 SEQ ID NO: 217 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 208 
                 TAS2R14 
                 NP_076411.1 
                 Receptor, 
                 Y121 
                 IANFSNSIFLyLKWRVKK 
                 SEQ ID NO: 218 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 209 
                 TAS2R8 
                 NP_076407.1 
                 Receptor, 
                 Y46 
                 KKKISTVDyILTNLVIAR 
                 SEQ ID NO: 219 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 210 
                 UNC93B1 
                 NP_12192.2 
                 Receptor, 
                 Y585 
                 RPCPyEQAQGGDGPEEQ 
                 SEQ ID NO: 220 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 211 
                 DDX3Y 
                 NP_004651.2 
                 RNA processing 
                 Y264 
                 KQyPISLVLAPTR 
                 SEQ ID NO: 221 
               
               
                   
               
               
                 212 
                 FUSIP1 
                 NP_006616.1 
                 RNA processing 
                 Y40 
                 YGPIVDVyVPLDFYTR 
                 SEQ ID NO: 222 
               
               
                   
               
               
                 213 
                 hnRNP G 
                 NP_002130.2 
                 RNA processing 
                 Y310 
                 yDDYSSSRDGYGGSRDSYSSSR 
                 SEQ ID NO: 223 
               
               
                   
               
               
                 214 
                 RBM25 
                 NP_067062.1 
                 RNA processing 
                 Y441 
                 DREEDEEDAyERR 
                 SEQ ID NO: 224 
               
               
                   
               
               
                 215 
                 RBM25 
                 NP_067062.1 
                 RNA processing 
                 Y457 
                 EKEAAyQERLK 
                 SEQ ID NO: 225 
               
               
                   
               
               
                 216 
                 RP11- 
                 CAI12730.1 
                 RNA processing 
                 Y258 
                 SSGSPyGGGYGSGGGSGGYGSR 
                 SEQ ID NO: 226 
               
               
                   
                 223F20.1 
               
               
                   
               
               
                 217 
                 RP11- 
                 CAI12730.1 
                 RNA processing 
                 Y262 
                 SSGSPYGGGyGSGGGSGGYGSR 
                 SEQ ID NO: 227 
               
               
                   
                 223F20.1 
               
               
                   
               
               
                 218 
                 RP11- 
                 CAI12730.1 
                 RNA processing 
                 Y271 
                 SSGSPYGGGYGSGGGSGGyGSR 
                 SEQ ID NO: 228 
               
               
                   
                 223F20.1 
               
               
                   
               
               
                 219 
                 SF3B1 
                 NP_036565.2 
                 RNA processing 
                 Y421 
                 VLPPPAGyVPIRTPARK 
                 SEQ ID NO: 229 
               
               
                   
               
               
                 220 
                 SFRS9 
                 NP_003760.1 
                 RNA processing 
                 Y160 
                 KEDMEyALR 
                 SEQ ID NO: 230 
               
               
                   
               
               
                 221 
                 SNRPD1 
                 NP_008869.1 
                 RNA processing 
                 Y67 
                 yFILPDSLPLDTLLVDVEPKVK 
                 SEQ ID NO: 231 
               
               
                   
               
               
                 222 
                 SRm300 
                 NP_057417.2 
                 RNA processing 
                 Y82 
                 CLELEEMMEEQGyEEQQIQEKVATFR 
                 SEQ ID NO: 232 
               
               
                   
               
               
                 223 
                 UPF3B 
                 NP_075386.1 
                 RNA processing 
                 Y98 
                 AyINFKNQEDIILFRDR 
                 SEQ ID NO: 233 
               
               
                   
               
               
                 224 
                 CCL17 
                 NP_002978.1 
                 Secreted protein 
                 Y37 
                 GTNVGRECCLEyFK 
                 SEQ ID NO: 234 
               
               
                   
               
               
                 225 
                 IGF1 
                 NP_000609.1 
                 Secreted protein 
                 Y151 
                 EVHLKNASRGSAGNKNyRM 
                 SEQ ID NO: 235 
               
               
                   
               
               
                 226 
                 LTF 
                 NP_002334.2 
                 Secreted protein 
                 Y547 
                 YYGyTGAFR 
                 SEQ ID NO: 236 
               
               
                   
               
               
                 227 
                 NPTX1 
                 NP_002513.2 
                 Secreted protein 
                 Y344 
                 DGVWEAYQDGTQGGSGENLAPyHPIK 
                 SEQ ID NO: 237 
               
               
                   
               
               
                 228 
                 PTGDS 
                 NP_000945.3 
                 Secreted protein 
                 Y149 
                 MATLySR 
                 SEQ ID NO: 239 
               
               
                   
               
               
                 229 
                 SCG3 
                 NP_037375.2 
                 Secreted protein 
                 Y208 
                 EANNyEEDPNK 
                 SEQ ID NO: 240 
               
               
                   
               
               
                 230 
                 calgizzarin 
                 NP_005611.1 
                 Transcriptional 
                 Y32 
                 YAGKDGYNyTLSK 
                 SEQ ID NO: 242 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 231 
                 IRF8 
                 NP_002154.1 
                 Transcriptional 
                 Y373 
                 LILVQIEQLyVR 
                 SEQ ID NO: 243 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 232 
                 JAZF1 
                 NP_778231.2 
                 Transcriptional 
                 Y55 
                 QELQQPTyVALSYINR 
                 SEQ ID NO: 244 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 233 
                 LDB2 
                 NP_001281.1 
                 Transcriptional 
                 Y203 
                 MGLTNFTLNyLR 
                 SEQ ID NO: 245 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 234 
                 N-CoR1 
                 NP_006302.2 
                 Transcriptional 
                 Y1488 
                 TPVSyQNTMSRGSPMMNRTSDVTISSNKST 
                 SEQ ID NO: 246 
               
               
                   
                   
                   
                 regulator 
                   
                 NHER 
               
               
                   
               
               
                 235 
                 NFkB-p105 
                 NP_003989.2 
                 Transcriptional 
                 Y60 
                 yVCEGPSHGGLPGASSEKNK 
                 SEQ ID NO: 247 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 236 
                 PRDM5 
                 NP_061169.2 
                 Transcriptional 
                 Y69 
                 GSKGEVLyILDATNPRHSNWLR 
                 SEQ ID NO: 249 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 237 
                 SALL1 
                 NP_002959.1 
                 Transcriptional 
                 Y753 
                 GNLKTHySVHRAMPPLR 
                 SEQ ID NO: 250 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 238 
                 SOX18 
                 NP_060889.1 
                 Transcriptional 
                 Y8 
                 MQRSPPGyGAQDDPPARR 
                 SEQ ID NO: 251 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 239 
                 TBX3 
                 NP_057653.3 
                 Transcriptional 
                 Y265 
                 FHIVRANDILKLPySTFR 
                 SEQ ID NO: 252 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 240 
                 ZBP-89 
                 NP_068799.1 
                 Transcriptional 
                 Y259 
                 THSGEKPYQCEyCLQYFSR 
                 SEQ ID NO: 253 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 241 
                 ZNF300 
                 NP_443092.1 
                 Transcriptional 
                 Y521 
                 IHTGEKPyICTECGKAFSQKSHLPGHQRIHT 
                 SEQ ID NO: 254 
               
               
                   
                   
                   
                 regulator 
                   
                 GEK 
               
               
                   
               
               
                 242 
                 ZNF406 
                 NP_065914.2 
                 Transcriptional 
                 Y916 
                 CSLCEyATRSKSNLK 
                 SEQ ID NO: 255 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 243 
                 ZNF431 
                 NP_597730-1 
                 Transcriptional 
                 Y29 
                 MDDLKYGVYPLKEASGCPGAERNLLVYSyF 
                 SEQ ID NO: 256 
               
               
                   
                   
                   
                 regulator 
                   
               
               
                   
               
               
                 244 
                 ZNF622 
                 NP_219482.1 
                 Transcnptional 
                 Y68 
                 AVAEEESKGSATyCTVCSK 
                 SEQ ID NO: 257 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 245 
                 ZNF630 
                 NP_001032824.1 
                 Transcriptional 
                 Y167 
                 IHTGEKPyVCGDCRK 
                 SEQ ID NO: 258 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 246 
                 EIF2B3 
                 NP_065098.1 
                 Translational 
                 Y264 
                 ELKSLDIySFIK 
                 SEQ ID NO: 259 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 247 
                 GCN1L1 
                 NP_006827.1 
                 Translational 
                 Y1205 
                 LMEIyQEK 
                 SEQ ID NO: 260 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 248 
                 FBW1B 
                 NP_036432.2 
                 Ubiquitin 
                 Y537 
                 TyTYISR 
                 SEQ ID NO: 261 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                 249 
                 FBXO9 
                 NP_036479.1 
                 Ubiquitin 
                 Y111 
                 AVEEEQNGALyEAIK 
                 SEQ ID NO: 262 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                 250 
                 UBR1 
                 NP_777576.1 
                 Ubiquitin 
                 Y1302 
                 EMVILFATTIyR 
                 SEQ ID NO: 263 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                 251 
                 USP10 
                 NP_005144.1 
                 Ubiquitin 
                 Y359 
                 SWASLFHDSKPSSSSPVAyVETK 
                 SEQ ID NO: 264 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                 252 
                 USP14 
                 NP_005142.1 
                 Ubiquitin 
                 Y285 
                 yLFTGLKLRLQEEITK 
                 SEQ ID NO: 265 
               
               
                   
                   
                   
                 conjugating 
               
               
                   
                   
                   
                 system 
               
               
                   
               
               
                 253 
                 ALS2CR8 
                 NP_079020.12 
                 Transcriptional 
                 Y330 
                 FPEyRVPTDPKIDK 
                 SEQ ID NO: 266 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 254 
                 ASB14 
                 NP_569058.1 
                 Unknown function 
                 Y216 
                 VMLDyVDQVR 
                 SEQ ID NO: 267 
               
               
                   
               
               
                 255 
                 ASB4 
                 NP_057200.1 
                 Unknown function 
                 Y426 
                 YLLLEPEGIIy 
                 SEQ ID NO: 268 
               
               
                   
               
               
                 256 
                 BNIP2 
                 NP_004321.1 
                 Adhesion or 
                 Y230 
                 MPSLGWLRKCyQQIDRR 
                 SEQ ID NO: 270 
               
               
                   
                   
                   
                 extracellular 
               
               
                   
                   
                   
                 matrix protein 
               
               
                   
               
               
                 257 
                 BTBD9 
                 NP_689946.1 
                 Unknown function 
                 Y318 
                 yIRIVGTHNVNK 
                 SEQ ID NO: 271 
               
               
                   
               
               
                 258 
                 C13orf10 
                 NP_071401.3 
                 Unknown function 
                 Y811 
                 yTELQLEAAK 
                 SEQ ID NO: 272 
               
               
                   
               
               
                 259 
                 C14orf145 
                 NP_689659.2 
                 Unknown function 
                 Y461 
                 yLSELQQSEALK 
                 SEQ ID NO: 274 
               
               
                   
               
               
                 260 
                 C14orf44 
                 NP_689658.1 
                 Unknown function 
                 Y532 
                 EyKKELEEMKQR 
                 SEQ ID NO: 275 
               
               
                   
               
               
                 261 
                 C17orf57 
                 NP_689560.2 
                 Unknown function 
                 Y319 
                 yKKKNSLSSKLPEPSISK 
                 SEQ ID NO: 276 
               
               
                   
               
               
                 262 
                 C17orf57 
                 NP_689560.2 
                 Unknown function 
                 Y345 
                 SNQyYSKIMENDDLESK 
                 SEQ ID NO: 277 
               
               
                   
               
               
                 263 
                 C17orf57 
                 NP_689560.2 
                 Unknown function 
                 Y346 
                 SNQYySKIMENDDLESK 
                 SEQ ID NO: 278 
               
               
                   
               
               
                 264 
                 C19orf15 
                 NP_067008.2 
                 Unknown function 
                 Y159 
                 ENFIYLADFPKELSIKyMAR 
                 SEQ ID NO: 279 
               
               
                   
               
               
                 265 
                 C6orf152 
                 NP_859065.1 
                 Unknown function 
                 Y265 
                 KRAyEAHDENKVLQK 
                 SEQ ID NO: 281 
               
               
                   
               
               
                 266 
                 CAP2 
                 NP_006357.1 
                 Cytoskeletal 
                 Y287 
                 HVTDDQKTyKNPSLR 
                 SEQ ID NO: 282 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 267 
                 CEP152 
                 NP_055800.1 
                 Unknown function 
                 Y175 
                 yLNHQLVIIK 
                 SEQ ID NO: 283 
               
               
                   
               
               
                 268 
                 CRIP1 
                 NP_001302.1 
                 Unknown function 
                 Y12 
                 CNKEVyFAER 
                 SEQ ID NO: 285 
               
               
                   
               
               
                 269 
                 CSRP1 
                 NP_004069.1 
                 Unknown function 
                 Y166 
                 GLESTTLADKDGEIyCKGCYAK 
                 SEQ ID NO: 286 
               
               
                   
               
               
                 270 
                 CSRP2 
                 NP_001312.1 
                 Unknown function 
                 Y66 
                 yGPKGYGYGQGAGTLNMDR 
                 SEQ ID NO: 287 
               
               
                   
               
               
                 271 
                 CSRP2 
                 NP_001312.1 
                 Unknown function 
                 Y71 
                 YGPKGyGYGQGAGTLNMDR 
                 SEQ ID NO: 288 
               
               
                   
               
               
                 272 
                 DCAKD 
                 NP_079095.2 
                 Unknown function 
                 Y109 
                 yVILDIPLLFETK 
                 SEQ ID NO: 289 
               
               
                   
               
               
                 273 
                 DKFZP781 
                 NP_689835.2 
                 Unknown function 
                 Y6 
                 MLVHDyDDER 
                 SEQ ID NO: 290 
               
               
                   
                 I1119 
               
               
                   
               
               
                 274 
                 DOCK5 
                 NP_079216.3 
                 Chromatin, DNA- 
                 Y1291 
                 DSYYVyTQQELK 
                 SEQ ID NO: 291 
               
               
                   
                   
                   
                 binding, DNA 
               
               
                   
                   
                   
                 repair or DNA 
               
               
                   
                   
                   
                 replication 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 275 
                 exophilin 5 
                 NP_055880.1 
                 Unknown function 
                 Y403 
                 yVYPRGFQENK 
                 SEQ ID NO: 292 
               
               
                   
               
               
                 276 
                 FLJ14235 
                 NP_060406.1 
                 Unknown function 
                 Y169 
                 EKLALALENEGyIK 
                 SEQ ID NO: 293 
               
               
                   
               
               
                 277 
                 FLJ20323 
                 NP_061878.2 
                 Unknown function 
                 Y665 
                 DGVDLMESyVDR 
                 SEQ ID NO: 294 
               
               
                   
               
               
                 278 
                 FLJ20758 
                 NP_060422.4 
                 Unknown function 
                 Y124 
                 SGENVAKFIINSyPK 
                 SEQ ID NO: 295 
               
               
                   
               
               
                 279 
                 FLJ46072 
                 NP_940890.2 
                 Unknown function 
                 Y458 
                 DQLYQQQyQWDPQLTPARPQGLFEK 
                 SEQ ID NO: 297 
               
               
                   
               
               
                 280 
                 GAGE3 
                 AAA82746.1 
                 Unknown function 
                 Y11 
                 STYyWPRPR 
                 SEQ ID NO: 299 
               
               
                   
               
               
                 281 
                 GAGE4 
                 NP_001465.1 
                 Unknown function 
                 Y10 
                 STyYWPRPR 
                 SEQ ID NO: 300 
               
               
                   
               
               
                 282 
                 GAGE5 
                 NP_001466.1 
                 Unknown function 
                 Y10 
                 STyYWPRPR 
                 SEQ ID NO: 301 
               
               
                   
               
               
                 283 
                 GAGE6 
                 NP_001467.1 
                 Unknown function 
                 Y10 
                 STyYWPRPR 
                 SEQ ID NO: 302 
               
               
                   
               
               
                 284 
                 GAGE7B 
                 NP_001468.1 
                 Unknown function 
                 Y10 
                 STyYWPRPR 
                 SEQ ID NO: 303 
               
               
                   
               
               
                 285 
                 GDAP1 
                 NP_061845.1 
                 Unknown function 
                 Y204 
                 yLKKILDELEK 
                 SEQ ID NO: 304 
               
               
                   
               
               
                 286 
                 HDGFRP3 
                 NP_057157.1 
                 Unknown function 
                 Y150 
                 SyTSKKSSKQSRK 
                 SEQ ID NO: 305 
               
               
                   
               
               
                 287 
                 HOXA4 
                 NP_002132.2 
                 Transcriptional 
                 Y210 
                 IHVSAVNPSyNGGEPK 
                 SEQ ID NO: 306 
               
               
                   
                   
                   
                 regulator 
               
               
                   
               
               
                 288 
                 KIAA0859 
                 NP_057019.3 
                 Unknown function 
                 Y282 
                 yTLHVVDSPTVKPSR 
                 SEQ ID NO: 307 
               
               
                   
               
               
                 289 
                 KIAA1196 
                 NP_065764.1 
                 Unknown function 
                 Y761 
                 GHVNCPNDCCEAIySSVSGLK 
                 SEQ ID NO: 308 
               
               
                   
               
               
                 290 
                 KIAA1279 
                 NP_056449.1 
                 Unknown function 
                 Y37 
                 ySARALLEEVK 
                 SEQ ID NO: 310 
               
               
                   
               
               
                 291 
                 KLHL15 
                 NP_085127.1 
                 Unknown function 
                 Y55 
                 ALLATQSDyFR 
                 SEQ ID NO: 311 
               
               
                   
               
               
                 292 
                 LRFN4 
                 NP_076941.2 
                 Receptor, 
                 Y594 
                 SCSLDLGDAGCyGYAR 
                 SEQ ID NO: 312 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 293 
                 MARVELD 
                 NP_653325.1 
                 Unknown function 
                 Y137 
                 SPLNSCKDPyGGSEGTFSSR 
                 SEQ ID NO: 313 
               
               
                   
                 2 
               
               
                   
               
               
                 294 
                 MLLT11 
                 NP_006809.1 
                 Unknown function 
                 Y9 
                 DPVSSQySSFLFWR 
                 SEQ ID NO: 314 
               
               
                   
               
               
                 295 
                 NHSL1 
                 XP_496826.1 
                 Unknown function 
                 Y103 
                 SSQYYSQGPTFAANASPFCDDyQDEDEET 
                 SEQ ID NO: 315 
               
               
                   
                   
                   
                   
                   
                 DQK 
               
               
                   
               
               
                 296 
                 PGM2 
                 NP_060760.2 
                 Enzyme, misc. 
                 Y568 
                 IKyYAELCAPPGNSDPEQLKK 
                 SEQ ID NO: 317 
               
               
                   
               
               
                 297 
                 PGM2L1 
                 NP_775853.2 
                 Enzyme, misc. 
                 Y159 
                 YVPTPFVPyAVQK 
                 SEQ ID NO: 318 
               
               
                   
               
               
                 298 
                 PLEKHB1 
                 NP_067023.1 
                 Receptor, 
                 Y175 
                 VYSPYQDyYEVVPPNAHEATYVR 
                 SEQ ID NO: 319 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 299 
                 PLEKHB1 
                 NP_067023.1 
                 Receptor, 
                 Y176 
                 VYSPYQDYyEVVPPNAHEATYVR 
                 SEQ ID NO: 320 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 300 
                 PLEKHB1 
                 NP_067023.1 
                 Receptor, 
                 Y188 
                 VYSPYQDYYEVVPPNAHEATyVR 
                 SEQ ID NO: 321 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 301 
                 PPIL4 
                 NP_624311.1 
                 Enzyme, misc. 
                 Y283 
                 TGESLCyAFIEFEKEEDCEK 
                 SEQ ID NO: 322 
               
               
                   
               
               
                 302 
                 R3HDM2 
                 NP_055740.2 
                 Unknown function 
                 Y591 
                 HSDLAALyTIVAVFPSPLAAQNASLR 
                 SEQ ID NO: 323 
               
               
                   
               
               
                 303 
                 RBM16 
                 NP_055707.3 
                 RNA processing 
                 Y17 
                 TFNSELYSLNDyKPPISK 
                 SEQ ID NO: 324 
               
               
                   
               
               
                 304 
                 RNF17 
                 NP_061911.2 
                 Unknown function 
                 Y398 
                 GSDTLWyRGKVMEVVGGAVR 
                 SEQ ID NO: 325 
               
               
                   
               
               
                 305 
                 SF4 
                 NP_066987.2 
                 RNA processing 
                 Y172 
                 QLPVAHRPSVFQSPDEDEEEDyEQWLEIK 
                 SEQ ID NO: 327 
               
               
                   
               
               
                 306 
                 SF4 
                 NP_357386.2 
                 RNA processing 
                 Y483 
                 AVQQHQHGyDSDEEVDSELGTWEHQLR 
                 SEQ ID NO: 328 
               
               
                   
               
               
                 307 
                 TSGA2 
                 NP_543136.1 
                 Cell cycle 
                 Y159 
                 yQGKFLNKNPVGPGK 
                 SEQ ID NO: 329 
               
               
                   
                   
                   
                 regulation 
               
               
                   
               
               
                 308 
                 VPS13D 
                 NP_056193.2 
                 Unknown function 
                 Y3391 
                 yIDTCMVIFAPR 
                 SEQ ID NO: 330 
               
               
                   
               
               
                 309 
                 WDR68 
                 NP_005819.2 
                 Unknown function 
                 Y94 
                 GVYPDLLATSGDyLR 
                 SEQ ID NO: 331 
               
               
                   
               
               
                 310 
                 ZNF512 
                 NP_115810.2 
                 Unknown function 
                 Y214 
                 yHVMANHNSLPILK 
                 SEQ ID NO: 332 
               
               
                   
               
               
                 311 
                 AP1G1 
                 NP_001119.3 
                 Vesicle protein 
                 Y326 
                 FLLNNDKNIRyVALTSLLK 
                 SEQ ID NO: 333 
               
               
                   
               
               
                 312 
                 EXOSC10 
                 NP_002676.1 
                 Vesicle protein 
                 Y530 
                 REDESYGyVLPNHMMLK 
                 SEQ ID NO: 334 
               
               
                   
               
               
                 313 
                 SCAMP4 
                 NP_524558.1 
                 Receptor, 
                 Y222 
                 EAQYNNFSGNSLPEYPTVPSyPGSGQWP 
                 SEQ ID NO: 335 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 314 
                 SEC31L1 
                 NP_055748.2 
                 Vesicle protein 
                 Y101 
                 MDSKGDVSGVLIAGGENGNIILyDPSK 
                 SEQ ID NO: 336 
               
               
                   
               
               
                 315 
                 SPRED2 
                 NP_861449.1 
                 Vesicle protein 
                 Y266 
                 GEVPKHDYNyPYVDSSDFGLGEDPK 
                 SEQ ID NO: 337 
               
               
                   
               
               
                 316 
                 SYTL4 
                 NP_542775.1 
                 Vesicle protein 
                 Y396 
                 KRSNPyVKTYLLPDK 
                 SEQ ID NO: 338 
               
               
                   
               
               
                 317 
                 TXLNA 
                 NP_787048.1 
                 Vesicle protein 
                 Y524 
                 VTEAPCyPGAPSTEASGQTGPQEPTSAR 
                 SEQ ID NO: 339 
               
               
                   
               
               
                 318 
                 UNC13A 
                 XP_038604.9 
                 Receptor, 
                 Y700 
                 TGSSDPyVTVQVGKTKKRTK 
                 SEQ ID NO: 340 
               
               
                   
                   
                   
                 channel, 
               
               
                   
                   
                   
                 transporter or 
               
               
                   
                   
                   
                 cell surface 
               
               
                   
                   
                   
                 protein 
               
               
                   
               
               
                 319 
                 VPS45A 
                 NP_009190.2 
                 Vesicle protein 
                 Y10 
                 QyISKMIEDSGPGMKVLLMDK 
                 SEQ ID NO: 341 
               
               
                   
               
            
           
         
       
     
     One of skill in the art will appreciate that, in many instances the utility of the instant invention is best understood in conjunction with an appreciation of the many biological roles and significance of the various target signaling proteins/polypeptides of the invention. The foregoing is illustrated in the following paragraphs summarizing the knowledge in the art relevant to a few non-limiting representative peptides containing selected phosphorylation sites according to the invention. 
     Beta actin, phosphorylated at Y53, is among the proteins listed in this patent. Beta actin is a highly conserved protein that is ubiquitously expressed at high levels in all eukaryotic cells. In vertebrates three main groups of actin isoforms, alpha, beta and gamma have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins coexist in all cell types as the dominant components of the cytoskeleton and are essential mediators of almost all processes of eukaryotic cells including cellular architecture, migration, adhesion, cell division, organelle transport, endocytosis, exocytosis, organogenesis, developmental migrations, brain patterning, secretion, etc. CST has made a fundamental discovery that beta actin has many sites of tyrosine phosphorylation. These include Tyr53, Tyr91, Tyr166, Tyr169, Tyr198, Tyr218, Tyr240, and Tyr294. We published the initial report of this discovery in 2004 (Rush J, et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 2004 Dec. 12). The only known site of beta actin phosphorylation reported by investigators outside of CST was made in 2007 for Thr203 (Villén J, Beausoleil S A, Gerber S A, Gygi S P Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci USA 2007 Jan. 30; 104(5): 1488-93). We believe that these phosphorylation sites are very important discoveries and that they are likely to regulate many of the above cellular processes mentioned above. Antibodies against these sites will be essential tools for investigating the role of specific sites of phosphorylation in regulating the cellular processes controlled by actin. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Melanoma (Anal Biochem 2001 Aug. 1; 295(1):17-21). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     RbBP1, phosphorylated at Y20, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Breast Neoplasms (Br J Cancer 1999 September; 81(2):342-9). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     CSPG2, phosphorylated at Y208, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Coronary Arteriosclerosis (Arterioscler Thromb Vasc Biol 2002 Oct. 1; 22(10):1642-8). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     FBN1, phosphorylated at Y2849, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Dissecting Aneurysm (Am J Hum Genet 1995 June; 56(6):1287-96). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     KIRREL, phosphorylated at Y574, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     cyclin B1, phosphorylated at Y177, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Non-Small-Cell Lung Carcinoma (Anticancer Res 2000 March-April; 20(2A):693-702). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     PITPNM2, phosphorylated at Y191, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     APCS, phosphorylated at Y142, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Arteriosclerosis (Arterioscler Thromb Vasc Biol 1995 February; 15(2):252-7). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     HSC70, phosphorylated at Y41, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Alzheimer Disease (Biochem Biophys Res Commun 2001 Jan. 12; 280(1):249-58). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     CK6, phosphorylated at Y551, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     moesin, phosphorylated at Y270, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Down Syndrome (Biochem Biophys Res Commun 2001 Sep. 7; 286(5):1191-4). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     supervillin, phosphorylated at Y120, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     CYP24A1, phosphorylated at Y220, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Prostatic Neoplasms (Endocrinology 1995 January; 136(1):20-6). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     OGT, phosphorylated at Y966, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Colorectal Neoplasms (Hum Mol Genet. 2001 Mar. 1; 10(5):513-8). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     SOS2, phosphorylated at Y679, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     PIK3R2, phosphorylated at Y74, is among the proteins listed in this patent. PIK3R2 is a regulatory subunit of phosphoinositide-3-kinase. PIK3R2 acts as an adapter, mediating the association of the p110 catalytic unit of the alpha, beta and delta enzymes to the plasma membrane, where p110 phosphorylates inositol lipids. The PI3K pathway is critical for insulin&#39;s actions in the liver in vivo (Cell Metab. 2006 May; 3(5):343-53). PIK3R2 mediates binding to a subset of tyrosine-phosphorylated proteins including the insulin receptor and the PDGFR through its SH2 domain (EMBO J. 1992 December; 11(12):4261-72). PIK3R2 plays a role in the regulation of chemotaxis (Eur J. Cell Biol. 2006 September; 85(9-10):873-95), and is necessary for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues (J Biol. Chem. 2003 Nov. 28; 278(48):48453-66). PIK3R2 is a potential therapeutic target in epithelial ovarian cancer (Clin Cancer Res. 2007 Sep. 15; 13(18):5314-21). Antibodies against PIK3R2 pY74 can be used to investigate the role of pY74 in critical cellular processes including diabetes, insulin-signaling, chemotaxis, and glycogen synthesis. 
     PIK4CA, phosphorylated at Y842, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     CENPE, phosphorylated at Y283, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     LRIG1, phosphorylated at Y941, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Neoplasms (Biochem Biophys Res Commun 2001 Jun. 29; 284(5):1155-61). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     ASK1, phosphorylated at Y814, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Ovarian Neoplasms (Endocrinology 2004 January; 145(1):49-58. Epub 2003 Sep. 18). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     LRRK2, phosphorylated at Y636, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Parkinson Disease (Am J Hum Genet. 2004 January; 74(1):11-9. Epub 2003 Dec. 19). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     PKR, phosphorylated at Y142, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Breast Neoplasms (Cancer Lett 2003 Jul. 10; 196(2):207-16). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     Fgr, phosphorylated at Y180, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Epstein-Barr Virus Infections (Mol Cell Biol 1991 March; 11(3):1500-7). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     Src, phosphorylated at Y439, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Adenocarcinoma, Pancreatic Neoplasms (Arch Biochem Biophys 2000 May 15; 377(2):350-6). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     EphA5, phosphorylated at Y790 and Y833, is among the proteins listed in this patent. (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     EphB1, phosphorylated at Y634, is among the proteins listed in this patent. EphB1 is a receptor tyrosine kinase of the Eph family. It is a receptor for ephrin-B proteins, which are cell-surface proteins that have a transmembrane and cytoplasmic domain. EphB1 binds ephrin-B proteins at sites of cell-cell contact, regulating the repulsion and adhesion of cells that underlie the establishment, maintenance, and remodeling of patterns of cellular organization. Eph signals are particularly important in regulating cell adhesion and cell migration during development, axon guidance, homeostasis and disease. Interactions between EphB receptor kinases and ephrin-B proteins transduce signals bidirectionally, signaling to both interacting cell types. Eph receptors and ephrins also regulate the adhesion of endothelial cells and are required for the remodeling of blood vessels. It has been reported that the activated form of EphB1 induces the phosphorylation of EphB1 Y594, which recruits the adapter protein NCK1 (J Biol Chem 1998 Jan. 16; 273(3): 1303-8); that the phosphorylation of Y600 and Y778 recruits the Src kinase and the adapter protein p52Shc to bind EphB1, promoting chemotaxis (J Cell Biol 2003 Aug. 18; 162(4): 661-71). The phosphorylation site of EphB1 pY634, herein defined as the amino acid residue that is phosphorylated+/−7 amino acids (AGEFGEVyKGRLKLP), is predicted to be phosphorylated by three kinases: PDGFR kinase, EGFR Kinase, and the Abl Kinase (Nucleic Acids Res. 2003 Jul. 1; 31(13):3635-41). All of these kinases play critical roles in the process of cell proliferation and oncogenesis (Nature. 2001 May 17; 411(6835):355-65). EphB1 pY634 is also predicted to recruit Fgr and PI3K-p85, the regulatory subunit of phosphoinositide-3-kinase, via interaction with their SI-12 domains. These predictions, made using the program Scan site (Nucleic Acids Res. 2003 Jul. 1; 31(13):3635-41), can be tested with an antibody against EphB1 pY634. Antibodies against EphB1 pY634 can be used to investigate the role of pY634 in regulating the repulsion and adhesion of cells that underlie the establishment of patterns of cellular organization, in regulating cell adhesion and cell migration during development, axon guidance, homeostasis and disease. Antibodies against EphB1 pY634 can be used to investigate the role of pY634 in neuropathic pain (Spine. 2007 Jul. 1; 32 (15):1592-8), Crohn&#39;s disease and epithelial wound healing (World J Gastroenterol. 2005 Jul. 14; 11(26):4024-31), growth and migration after stroke (Exp Neurol. 2005 June; 193(2):291-311), and regulating cell adhesion and cell migration during development, and axon guidance. 
     HER4, phosphorylated at Y1128, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Non-Small-Cell Lung Carcinoma (Anticancer Res 1999 January-February; 19(1A):481-6). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     Cx43, phosphorylated at Y267, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Eye Abnormalities (Am J Hum Genet 2003 February; 72(2):408-18). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     NFkB-p105, phosphorylated at Y60, is among the proteins listed in this patent. This protein has potential diagnostic and/or therapeutic implications based on association with the following diseases: Prostatic Neoplasms (Anticancer Res 2003 September-October; 23(5A):3855-61). (PhosphoSite®, Cell Signaling Technology (Danvers, Mass.), Human PSD™, Biobase Corporation, (Beverly, Mass.)). 
     The invention also provides peptides comprising a novel phosphorylation site of the invention. In one particular embodiment, the peptides comprise any one of the an amino acid sequences as set forth in column E of Table 1 and  FIG. 2 , which are trypsin-digested peptide fragments of the parent proteins. Alternatively, a parent signaling protein listed in Table 1 may be digested with another protease, and the sequence of a peptide fragment comprising a phosphorylation site can be obtained in a similar way. Suitable proteases include, but are not limited to, serine proteases (e.g. hepsin), metallo proteases (e.g. PUMP 1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc. 
     The invention also provides proteins and peptides that are mutated to eliminate a novel phosphorylation site of the invention. Such proteins and peptides are particular useful as research tools to understand complex signaling transduction pathways of cancer cells, for example, to identify new upstream kinase(s) or phosphatase(s) or other proteins that regulates the activity of a signaling protein; to identify downstream effector molecules that interact with a signaling protein, etc. 
     Various methods that are well known in the art can be used to eliminate a phosphorylation site. For example, the phosphorylatable tyrosine may be mutated into a non-phosphorylatable residue, such as phenylalanine. A “phosphorylatable” amino acid refers to an amino acid that is capable of being modified by addition of a phosphate group (any includes both phosphorylated form and unphosphorylated form). Alternatively, the tyrosine may be deleted. Residues other than the tyrosine may also be modified (e.g., delete or mutated) if such modification inhibits the phosphorylation of the tyrosine residue. For example, residues flanking the tyrosine may be deleted or mutated, so that a kinase can not recognize/phosphorylate the mutated protein or the peptide. Standard mutagenesis and molecular cloning techniques can be used to create amino acid substitutions or deletions. 
     2. Modulators of the Phosphorylation Sites 
     In another aspect, the invention provides a modulator that modulates tyrosine phosphorylation at a novel phosphorylation site of the invention, including small molecules, peptides comprising a novel phosphorylation site, and binding molecules that specifically bind at a novel phosphorylation site, including but not limited to antibodies or antigen-binding fragments thereof. 
     Modulators of a phosphorylation site include any molecules that directly or indirectly counteract, reduce, antagonize or inhibit tyrosine phosphorylation of the site. The modulators may compete or block the binding of the phosphorylation site to its upstream kinase(s) or phosphatase(s), or to its downstream signaling transduction molecule(s). 
     The modulators may directly interact with a phosphorylation site. The modulator may also be a molecule that does not directly interact with a phosphorylation site. For example, the modulators can be dominant negative mutants, i.e., proteins and peptides that are mutated to eliminate the phosphorylation site. Such mutated proteins or peptides could retain the binding ability to a downstream signaling molecule but lose the ability to trigger downstream signaling transduction of the wild type parent signaling protein. 
     The modulators include small molecules that modulate the tyrosine phosphorylation at a novel phosphorylation site of the invention. Chemical agents, referred to in the art as “small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, less than 5,000, less than 1,000, or less than 500 daltons. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of a phosphorylation site of the invention or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries. Methods for generating and obtaining compounds are well known in the art (Schreiber S L, Science 151: 1964-1969 (2000); Radmann J. and Gunther J., Science 151: 1947-1948 (2000)). 
     The modulators also include peptidomimetics, small protein-like chains designed to mimic peptides. Peptidomimetics may be analogues of a peptide comprising a phosphorylation site of the invention. Peptidomimetics may also be analogues of a modified peptide that are mutated to eliminate a phosphorylation site of the invention. Peptidomimetics (both peptide and non-peptidyl analogues) may have improved properties (e.g., decreased proteolysis, increased retention or increased bioavailability). Peptidomimetics generally have improved oral availability, which makes them especially suited to treatment of disorders in a human or animal. 
     In certain embodiments, the modulators are peptides comprising a novel phosphorylation site of the invention. In certain embodiments, the modulators are antibodies or antigen-binding fragments thereof that specifically bind at a novel phosphorylation site of the invention. 
     3. Heavy-Isotope Labeled Peptides (AQUA Peptides). 
     In another aspect, the invention provides peptides comprising a novel phosphorylation site of the invention. In a particular embodiment, the invention provides Heavy-Isotype Labeled Peptides (AQUA peptides) comprising a novel phosphorylation site. Such peptides are useful to generate phosphorylation site-specific antibodies for a novel phosphorylation site. Such peptides are also useful as potential diagnostic tools for screening carcinoma, or as potential therapeutic agents for treating carcinoma. 
     The peptides may be of any length, typically six to fifteen amino acids. The novel tyrosine phosphorylation site can occur at any position in the peptide; if the peptide will be used as an immunogen, it preferably is from seven to twenty amino acids in length. In some embodiments, the peptide is labeled with a detectable marker. 
     “Heavy-isotope labeled peptide” (used interchangeably with AQUA peptide) refers to a peptide comprising at least one heavy-isotope label, as described in WO/03016861, “Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry” (Gygi et al.) (the teachings of which are hereby incorporated herein by reference, in their entirety). The amino acid sequence of an AQUA peptide is identical to the sequence of a proteolytic fragment of the parent protein in which the novel phosphorylation site occurs. AQUA peptides of the invention are highly useful for detecting, quantitating or modulating a phosphorylation site of the invention (both in phosphorylated and unphosphorylated forms) in a biological sample. 
     A peptide of the invention, including an AQUA peptides comprises any novel phosphorylation site. Preferably, the peptide or AQUA peptide comprises a novel phosphorylation site of a protein in Table 1 that is a g protein or regulator protein, adhesion or extra-cellular matrix proteins, cytoskeletal proteins, enzyme proteins, kinases (non-protein), motor or contractile proteins, protein kinases, receptor/channel/transporter/cell surface proteins, RNA binding proteins and transcriptional regulators. 
     Particularly preferred peptides and AQUA peptides are these comprising a novel tyrosine phosphorylation site (shown as a lower case “y” in a sequence listed in Table 1) selected from the group consisting of SEQ ID NOs: 110 (SOS2); 17 (DLL1); 54 (actin, alpha 1); 55 (actin, beta); 78 (radixin); 86 (AKR1C1); 100 (OGT); 115 (PI4KII); 117 (PIK4CA); 131 (KIF5B); 154 (DYRK4); 167 (Fgr); 168 (Fyn); 170 (Src); 172 (Yes); 173 (Yes); 174 (Yes); 176 (EphA5); 180 (AQP1); 183 (ATP6VOA1); 189 (Cx43); 192 (GABRB2); 203 (Nup153); 211 (SLC25A4); 231 (SNRPD1); 246 (NCoR1); 247 (NFkB-p105); 260 (GCN1L1); 9 (VANGL1); 44 (NDN); 53 (NPAS3); 118 (ephrin-B1); 140 (MTM1); 265 (USP14); 286 (CSRP1); 314 (MLLT11); 322 (PPIL4); 334 (EXOSC10) and; 336 (SEC31 L1). 
     In some embodiments, the peptide or AQUA peptide comprises the amino acid sequence shown in any one of the above listed SEQ ID NOs. In some embodiments, the peptide or AQUA peptide consists of the amino acid sequence in said SEQ ID NOs. In some embodiments, the peptide or AQUA peptide comprises a fragment of the amino acid sequence in said SEQ ID NOs., wherein the fragment is six to twenty amino acid long and includes the phosphorylatable tyrosine. In some embodiments, the peptide or AQUA peptide consists of a fragment of the amino acid sequence in said SEQ ID NOs., wherein the fragment is six to twenty amino acid long and includes the phosphorylatable tyrosine. 
     In certain embodiments, the peptide or AQUA peptide comprises any one of the SEQ ID NOs listed in column H, which are trypsin-digested peptide fragments of the parent proteins. 
     It is understood that parent protein listed in Table 1 may be digested with any suitable protease (e.g., serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc), and the resulting peptide sequence comprising a phosphorylated site of the invention may differ from that of trypsin-digested fragments (as set forth in Column E), depending the cleavage site of a particular enzyme. An AQUA peptide for a particular a parent protein sequence should be chosen based on the amino acid sequence of the parent protein and the particular protease for digestion; that is, the AQUA peptide should match the amino acid sequence of a proteolytic fragment of the parent protein in which the novel phosphorylation site occurs. 
     An AQUA peptide is preferably at least about 6 amino acids long. The preferred ranged is about 7 to 15 amino acids. 
     The AQUA method detects and quantifies a target protein in a sample by introducing 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. By comparing to the peptide standard, one may readily determines the quantity of a peptide having the same sequence and protein modification(s) in the biological sample. Briefly, the AQUA methodology has two stages: (1) peptide internal standard selection and validation; method development; and (2) implementation using validated peptide internal standards to detect and quantify a target protein in a 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 used, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify 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 a particular protease for digestion. The peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes ( 13 C,  15 N). 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 the modified form of the 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 may be developed for a known phosphorylation site previously identified by the IAP-LC-MS/MS method within a target protein. One AQUA peptide incorporating the phosphorylated form of the site, and a second AQUA peptide incorporating the unphosphorylated form of site may be developed. In this way, the two standards may be used to detect and quantify both the phosphorylated and unphosphorylated 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. PUMP 1), 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 is outside a phosphorylation site may be selected as internal standard to determine the quantity of all forms of the target protein. Alternatively, a peptide encompassing a phosphorylated site may be selected as internal standard to detect and quantify only the phosphorylated form of the target protein. Peptide standards for both phosphorylated form and unphosphorylated form can be used together, to determine the extent of phosphorylation in a particular 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  13 C,  15 N,  17 O,  18 O, or  34 S, 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 (MS n ) 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 MS 3  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 used. 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 MS n  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&#39;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. 
     Accordingly, AQUA internal peptide standards (heavy-isotope labeled peptides) may be produced, as described above, for any of the 318 novel phosphorylation sites of the invention (see Table 1/ FIG. 2 ). For example, peptide standards for a given phosphorylation site (e.g., an AQUA peptide having the sequence AAEDAVyELQSK (SEQ ID NO: 1), wherein “y” corresponds to phosphorylatable tyrosine 1352 of envoplakin) may be produced for both the phosphorylated and unphosphorylated forms of the sequence. Such standards may be used to detect and quantify both phosphorylated form and unphosphorylated form of the parent signaling protein (e.g., envoplakin) in a biological sample. 
     Heavy-isotope labeled equivalents of a phosphorylation site of the invention, 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. 
     The novel phosphorylation sites of the invention 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 (e.g., 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) that may be used for detecting, quantitating, or modulating any of the phosphorylation sites of the invention (Table 1). For example, an AQUA peptide having the sequence YPAVDyNLVQDLK (SEQ ID NO: 17), wherein y (Tyr 641) may be either phosphotyrosine or tyrosine, and wherein V=labeled valine (e.g.,  4 C)) is provided for the quantification of phosphorylated (or unphosphorylated) form of DLL 1 (an ahesion or extracellular matrix protein) in a biological sample. 
     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, AQUA peptides corresponding to both the phosphorylated and unphosphorylated forms of SEQ ID NO: 17 (a trypsin-digested fragment of DLL1, with a tyrosine 641 phosphorylation site) may be used to quantify the amount of phosphorylated DLL1 in a biological sample, e.g., a tumor cell sample or a sample before or after treatment with a therapeutic agent. 
     Peptides and AQUA peptides provided by the invention will be highly useful in the further study of signal transduction anomalies underlying cancer, including carcinomas. Peptides and AQUA peptides of the invention may also be used for identifying diagnostic/bio-markers of carcinomas, identifying new potential drug targets, and/or monitoring the effects of test therapeutic agents on signaling proteins and pathways. 
     4. Phosphorylation Site-Specific Antibodies 
     In another aspect, the invention discloses phosphorylation site-specific binding molecules that specifically bind at a novel tyrosine phosphorylation site of the invention, and that distinguish between the phosphorylated and unphosphorylated forms. In one embodiment, the binding molecule is an antibody or an antigen-binding fragment thereof. The antibody may specifically bind to an amino acid sequence comprising a phosphorylation site identified in Table 1. 
     In some embodiments, the antibody or antigen-binding fragment thereof specifically binds the phosphorylated site. In other embodiments, the antibody or antigen-binding fragment thereof specially binds the unphosphorylated site. An antibody or antigen-binding fragment thereof specially binds an amino acid sequence comprising a novel tyrosine phosphorylation site in Table 1 when it does not significantly bind any other site in the parent protein and does not significantly bind a protein other than the parent protein. An antibody of the invention is sometimes referred to herein as a “phospho-specific” antibody. 
     An antibody or antigen-binding fragment thereof specially binds an antigen when the dissociation constant is ≦1 mM, preferably ≦100 nM, and more preferably ≦10 nM. 
     In some embodiments, the antibody or antigen-binding fragment of the invention binds an amino acid sequence that comprises a novel phosphorylation site of a protein in Table 1 that is a g protein or regulator protein, adhesion or extra-cellular matrix protein, cytoskeletal protein, enzyme protein, kinases (non-protein), motor or contractile protein, protein kinase, receptor/channel/transporter/cell surface protein, RNA binding protein or a transcriptional regulator. 
     In particularly preferred embodiments, an antibody or antigen-binding fragment thereof of the invention specially binds an amino acid sequence comprising a novel tyrosine phosphorylation site shown as a lower case “y” in a sequence listed in Table 1 selected from the group consisting of SEQ ID NOS: 110 (SOS2); 17 (DLL1); 54 (actin, alpha 1); 55 (actin, beta); 78 (radixin); 86 (AKR1C1); 100 (OGT); 115 (PI4KII); 117 (PIK4CA); 131 (KIF5B); 154 (DYRK4); 167 (Fgr); 168 (Fyn); 170 (Src); 172 (Yes); 173 (Yes); 174 (Yes); 176 (EphA5); 180 (AQP1); 183 (ATP6VOA1); 189 (Cx43); 192 (GABRB2); 203 (Nup153); 211 (SLC25A4); 231 (SNRPD1); 246 (NCoR1); 247 (NFkB-p105); 260 (GCN1L1); 9 (VANGL1); 44 (NDN); 53 (NPAS3); 118 (ephrin-B1); 140 (MTM1); 265 (USP14); 286 (CSRP1); 314 (MLLT11); 322 (PPIL4); 334 (EXOSC10) and; 336 (SEC31L1). 
     In some embodiments, an antibody or antigen-binding fragment thereof of the invention specifically binds an amino acid sequence comprising any one of the above listed SEQ ID NOs. In some embodiments, an antibody or antigen-binding fragment thereof of the invention especially binds an amino acid sequence comprises a fragment of one of said SEQ ID NOs., wherein the fragment is four to twenty amino acid long and includes the phosphorylatable tyrosine. 
     In certain embodiments, an antibody or antigen-binding fragment thereof of the invention specially binds an amino acid sequence that comprises a peptide produced by proteolysis of the parent protein with a protease wherein said peptide comprises a novel tyrosine phosphorylation site of the invention. In some embodiments, the peptides are produced from trypsin digestion of the parent protein. The parent protein comprising the novel tyrosine phosphorylation site can be from any species, preferably from a mammal including but not limited to non-human primates, rabbits, mice, rats, goats, cows, sheep, and guinea pigs. In some embodiments, the parent protein is a human protein and the antibody binds an epitope comprising the novel tyrosine phosphorylation site shown by a lower case “y” in Column E of Table 1. Such peptides include any one of the SEQ ID NOs. 
     An antibody of the invention can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgG, IgA or IgD or sub-isotype including IgG1, IgG2, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. 
     Also within the invention are antibody molecules with fewer than 4 chains, including single chain antibodies, Camelid antibodies and the like and components of the antibody, including a heavy chain or a light chain. The term “antibody” (or “antibodies”) refers to all types of immunoglobulins. The term “an antigen-binding fragment of an antibody” refers to any portion of an antibody that retains specific binding of the intact antibody. An exemplary antigen-binding fragment of an antibody is the heavy chain and/or light chain CDR, or the heavy and/or light chain variable region. The term “does not bind,” when appeared in context of an antibody&#39;s binding to one phospho-form (e.g., phosphorylated form) of a sequence, means that the antibody does not substantially react with the other phospho-form (e.g., non-phosphorylated form) of the same sequence. One of skill in the art will appreciate that the expression may be applicable in those instances when (1) a phospho-specific antibody either does not apparently bind to the non-phospho form of the antigen as ascertained in commonly used experimental detection systems (Western blotting, IHC, Immunofluorescence, etc.); (2) where there is some reactivity with the surrounding amino acid sequence, but that the phosphorylated residue is an immunodominant feature of the reaction. In cases such as these, there is an apparent difference in affinities for the two sequences. Dilutional analyses of such antibodies indicates that the antibodies apparent affinity for the phosphorylated form is at least 10-100 fold higher than for the non-phosphorylated form; or where (3) the phospho-specific antibody reacts no more than an appropriate control antibody would react under identical experimental conditions. A control antibody preparation might be, for instance, purified immunoglobulin from a pre-immune animal of the same species, an isotype- and species-matched monoclonal antibody. Tests using control antibodies to demonstrate specificity are recognized by one of skill in the art as appropriate and definitive. 
     In some embodiments an immunoglobulin chain may comprise in order from 5′ to 3′, a variable region and a constant region. The variable region may comprise three complementarity determining regions (CDRs), with interspersed framework (FR) regions for a structure FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Also within the invention are heavy or light chain variable regions, framework regions and CDRs. An antibody of the invention may comprise a heavy chain constant region that comprises some or all of a CH1 region, hinge, CH2 and CH3 region. 
     An antibody of the invention may have an binding affinity (K D ) of 1×10 −7 M or less. In other embodiments, the antibody binds with a K D  of 1×10 −8  M, 1×10 −9  M, 1×10 −1 ° M, 1×10 −11 M, 1×10 −12 M or less. In certain embodiments, the K D  is 1 pM to 500 pM, between 500 pM to 1 μM, between 1 μM to 100 nM, or between 100 mM to 10 nM. 
     Antibodies of the invention can be derived from any species of animal, preferably a mammal. Non-limiting exemplary natural antibodies include antibodies derived from human, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al., WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al., WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated by reference in their entirety). Natural antibodies are the antibodies produced by a host animal. “Genetically altered antibodies” refer to antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics. 
     The antibodies of the invention include antibodies of any isotype including IgM, IgG, IgD, IgA and IgE, and any sub-isotype, including IgG1, IgG2a, IgG2b, IgG3 and IgG4, IgE1, IgE2 etc. The light chains of the antibodies can either be kappa light chains or lambda light chains. 
     Antibodies disclosed in the invention may be polyclonal or monoclonal. As used herein, the term “epitope” refers to the smallest portion of a protein capable of selectively binding to the antigen binding site of an antibody. It is well accepted by those skilled in the art that the minimal size of a protein epitope capable of selectively binding to the antigen binding site of an antibody is about five or six to seven amino acids. 
     Other antibodies specifically contemplated are oligoclonal antibodies. As used herein, the phrase “oligoclonal antibodies” refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule. In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixtures of antibodies that are applicable for an intended purpose and desired need. 
     Recombinant antibodies against the phosphorylation sites identified in the invention are also included in the present application. These recombinant antibodies have the same amino acid sequence as the natural antibodies or have altered amino acid sequences of the natural antibodies in the present application. They can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., Dower et al., WO91/17271 and McCafferty et al., WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated by reference in their entirety). 
     Antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab′) 2  fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203. 
     The genetically altered antibodies should be functionally equivalent to the above-mentioned natural antibodies. In certain embodiments, modified antibodies provide improved stability or/and therapeutic efficacy. Examples of modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies of this application can be modified post-translationally (e.g., acetylation, and/or phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group). 
     Antibodies with engineered or variant constant or Fc regions can be useful in modulating effector functions, such as, for example, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such antibodies with engineered or variant constant or Fc regions may be useful in instances where a parent singling protein (Table 1) is expressed in normal tissue; variant antibodies without effector function in these instances may elicit the desired therapeutic response while not damaging normal tissue. Accordingly, certain aspects and methods of the present disclosure relate to antibodies with altered effector functions that comprise one or more amino acid substitutions, insertions, and/or deletions. 
     In certain embodiments, genetically altered antibodies are chimeric antibodies and humanized antibodies. 
     The chimeric antibody is an antibody having portions derived from different antibodies. For example, a chimeric antibody may have a variable region and a constant region derived from two different antibodies. The donor antibodies may be from different species. In certain embodiments, the variable region of a chimeric antibody is non-human, e.g., murine, and the constant region is human. 
     The genetically altered antibodies used in the invention include CDR grafted humanized antibodies. In one embodiment, the humanized antibody comprises heavy and/or light chain CDRs of a non-human donor immunoglobulin and heavy chain and light chain frameworks and constant regions of a human acceptor immunoglobulin. The method of making humanized antibody is disclosed in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of which is incorporated herein by reference in its entirety. 
     Antigen-binding fragments of the antibodies of the invention, which retain the binding specificity of the intact antibody, are also included in the invention. Examples of these antigen-binding fragments include, but are not limited to, partial or full heavy chains or light chains, variable regions, or CDR regions of any phosphorylation site-specific antibodies described herein. 
     In one embodiment of the application, the antibody fragments are truncated chains (truncated at the carboxyl end). In certain embodiments, these truncated chains possess one or more immunoglobulin activities (e.g., complement fixation activity). Examples of truncated chains include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CH1 domains); Fd fragments (consisting of the VH and CH1 domains); Fv fragments (consisting of VL and VH domains of a single chain of an antibody); dAb fragments (consisting of a VH domain); isolated CDR regions; (Fab′) 2  fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region). The truncated chains can be produced by conventional biochemical techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art. These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CH1 to produce Fab fragments or after the hinge region to produce (Fab′) 2  fragments. Single chain antibodies may be produced by joining VL- and VH-coding regions with a DNA that encodes a peptide linker connecting the VL and VH protein fragments 
     Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment of an antibody yields an F(ab′) 2  fragment that has two antigen-combining sites and is still capable of cross-linking antigen. 
     “Fv” usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L  dimer. Collectively, the CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site. 
     Thus, in certain embodiments, the antibodies of the application may comprise 1, 2, 3, 4, 5, 6, or more CDRs that recognize the phosphorylation sites identified in Column E of Table 1. 
     The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′) 2  antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. 
     “Single-chain Fv” or “scFv” antibody fragments comprise the V H  and V L  domains of an antibody, wherein these domains are present in a single polypeptide chain. In certain embodiments, the Fv polypeptide further comprises a polypeptide linker between the V H  and V L  domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315. 
     SMIPs are a class of single-chain peptides engineered to include a target binding region and effector domain (CH2 and CH3 domains). See, e.g., U.S. Patent Application Publication No. 20050238646. The target binding region may be derived from the variable region or CDRs of an antibody, e.g., a phosphorylation site-specific antibody of the application. Alternatively, the target binding region is derived from a protein that binds a phosphorylation site. 
     Bispecific antibodies may be monoclonal, human or humanized antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the phosphorylation site, the other one is for any other antigen, such as for example, a cell-surface protein or receptor or receptor subunit. Alternatively, a therapeutic agent may be placed on one arm. The therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc. 
     In some embodiments, the antigen-binding fragment can be a diabody. The term “diabody” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). 
     Camelid antibodies refer to a unique type of antibodies that are devoid of light chain, initially discovered from animals of the camelid family. The heavy chains of these so-called heavy-chain antibodies bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain, referred to as VHH. VHHs show homology with the variable domain of heavy chains of the human VHIII family. The VHHs obtained from an immunized camel, dromedary, or llama have a number of advantages, such as effective production in microorganisms such as  Saccharomyces cerevisiae.    
     In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276 B1; U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 B1. See also, Newman et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird et al., Science, 242: 423-426 (1988)), regarding single chain antibodies. 
     In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. 
     Since the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins or conjugates having novel properties. 
     Non-immunoglobulin binding polypeptides are also contemplated. For example, CDRs from an antibody disclosed herein may be inserted into a suitable non-immunoglobulin scaffold to create a non-immunoglobulin binding polypeptide. Suitable candidate scaffold structures may be derived from, for example, members of fibronectin type III and cadherin superfamilies. 
     Also contemplated are other equivalent non-antibody molecules, such as protein binding domains or aptamers, which bind, in a phospho-specific manner, to an amino acid sequence comprising a novel phosphorylation site of the invention. See, e.g., Neuberger et al., Nature 312: 604 (1984). Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. DNA or RNA aptamers are typically short oligonucleotides, engineered through repeated rounds of selection to bind to a molecular target. Peptide aptamers typically consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint generally increases the binding affinity of the peptide aptamer to levels comparable to an antibody (nanomolar range). 
     The invention also discloses the use of the phosphorylation site-specific antibodies with immunotoxins. Conjugates that are immunotoxins including antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. In certain embodiments, antibody conjugates may comprise stable linkers and may release cytotoxic agents inside cells (see U.S. Pat. Nos. 6,867,007 and 6,884,869). The conjugates of the present application can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers et al., Seminars Cell Biol 2:59-70 (1991) and by Fanger et al., Immunol Today 12:51-54 (1991). Exemplary immunotoxins include radiotherapeutic agents, ribosome-inactivating proteins (RIPs), chemotherapeutic agents, toxic peptides, or toxic proteins. 
     The phosphorylation site-specific antibodies disclosed in the invention may be used singly or in combination. The antibodies may also be used in an array format for high throughput uses. An antibody microarray is a collection of immobolized antibodies, typically spotted and fixed on a solid surface (such as glass, plastic and silicon chip). 
     In another aspect, the antibodies of the invention modulate at least one, or all, biological activities of a parent protein identified in Column A of Table 1. The biological activities of a parent protein identified in Column A of Table 1 include: 1) ligand binding activities (for instance, these neutralizing antibodies may be capable of competing with or completely blocking the binding of a parent signaling protein to at least one, or all, of its ligands; 2) signaling transduction activities, such as receptor dimerization, or tyrosine phosphorylation; and 3) cellular responses induced by a parent signaling protein, such as oncogenic activities (e.g., cancer cell proliferation mediated by a parent signaling protein), and/or angiogenic activities. 
     In certain embodiments, the antibodies of the invention may have at least one activity selected from the group consisting of: 1) inhibiting cancer cell growth or proliferation; 2) inhibiting cancer cell survival; 3) inhibiting angiogenesis; 4) inhibiting cancer cell metastasis, adhesion, migration or invasion; 5) inducing apoptosis of cancer cells; 6) incorporating a toxic conjugate; and 7) acting as a diagnostic marker. 
     In certain embodiments, the phosphorylation site specific antibodies disclosed in the invention are especially indicated for diagnostic and therapeutic applications as described herein. Accordingly, the antibodies may be used in therapies, including combination therapies, in the diagnosis and prognosis of disease, as well as in the monitoring of disease progression. The invention, thus, further includes compositions comprising one or more embodiments of an antibody or an antigen binding portion of the invention as described herein. The composition may further comprise a pharmaceutically acceptable carrier. The composition may comprise two or more antibodies or antigen-binding portions, each with specificity for a different novel tyrosine phosphorylation site of the invention or two or more different antibodies or antigen-binding portions all of which are specific for the same novel tyrosine phosphorylation site of the invention. A composition of the invention may comprise one or more antibodies or antigen-binding portions of the invention and one or more additional reagents, diagnostic agents or therapeutic agents. 
     The present application provides for the polynucleotide molecules encoding the antibodies and antibody fragments and their analogs described herein. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each antibody amino acid sequence. The desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide. In one embodiment, the codons that are used comprise those that are typical for human or mouse (see, e.g., Nakamura, Y., Nucleic Acids Res. 28: 292 (2000)). 
     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 targeted 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., A NTIBODY  E NGINEERING  P ROTOCOLS , 1995, Humana Press, Sudhir Paul editor.) 
     5. Methods of Making Phosphorylation site-Specific Antibodies 
     In another aspect, the invention provides a method for making phosphorylation site-specific antibodies. 
     Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen comprising a novel tyrosine phosphorylation site of the invention. (i.e. a phosphorylation site shown in Table 1) in either the phosphorylated or unphosphorylated state, depending upon the desired specificity of the antibody, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures and screening and isolating a polyclonal antibody specific for the novel tyrosine phosphorylation site of interest as further described below. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane,  Antibodies: A Laboratory Manual , New York: Cold Spring Harbor Press, 1990. 
     The immunogen may be the full length protein or a peptide comprising the novel tyrosine phosphorylation site of interest. In some embodiments the immunogen is a peptide of from 7 to 20 amino acids in length, preferably about 8 to 17 amino acids in length. In some embodiments, the peptide antigen desirably will comprise about 3 to 8 amino acids on each side of the phosphorylatable tyrosine. In yet other embodiments, the peptide antigen desirably will comprise four or more amino acids flanking each side of the phosphorylatable amino acid and encompassing it. 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 &amp; Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik,  Methods In Enzymology,  201: 264-283 (1991); Merrifield,  J. Am. Chem. Soc.  85: 21-49 (1962)). 
     Suitable peptide antigens may comprise all or partial sequence of a trypsin-digested fragment as set forth in Column E of Table 1/ FIG. 2 . Suitable peptide antigens may also comprise all or partial sequence of a peptide fragment produced by another protease digestion. 
     Preferred immunogens are those that comprise a novel phosphorylation site of a protein in Table 1 that is a g protein or regulator protein, adhesion or extra-cellular matrix protein, cytoskeletal protein, enzyme protein, kinases (non-protein), motor or contractile protein, protein kinase, receptor/channel/transporter/cell surface protein, RNA binding protein or a transcriptional regulator. In some embodiments, the peptide immunogen is an AQUA peptide, for example, any one of SEQ ID NOS: 1, 3-7, 9-46, 48, 50-60, 62-65, 67-73, 75-95, 97-168, 170-209, 211, 213-237, 239-240, 242-247, 249-268, 270-272, 274-279, 281-283, 285-295, 297, 299-308, 310-315, 317-325, 327-341. 
     Particularly preferred immunogens are peptides comprising any one of the novel tyrosine phosphorylation site shown as a lower case “y” in a sequence listed in Table 1 selected from the group consisting of SEQ ID NOS: 110 (SOS2); 17 (DLL1); 54 (actin, alpha 1); 55 (actin, beta); 78 (radixin); 86 (AKR1C1); 100 (OGT); 115 (PI4KII); 117 (PIK4CA); 131 (KIF5B); 154 (DYRK4); 167 (Fgr); 168 (Fyn); 170 (Src); 172 (Yes); 173 (Yes); 174 (Yes); 176 (EphA5); 180 (AQP1); 183 (ATP6VOA1); 189 (Cx43); 192 (GABRB2); 203 (Nup153); 211 (SLC25A4); 231 (SNRPD1); 246 (NCoR1); 247 (NFkB-p105); 260 (GCN1L1); 9 (VANGL1); 44 (NDN); 53 (NPAS3); 118 (ephrin-B1); 140 (MTM1); 265 (USP14); 286 (CSRP1); 314 (MLLT11); 322 (PPIL4); 334 (EXOSC10) and; 336 (SEC31L1). 
     In some embodiments the immunogen is administered with an adjuvant. Suitable adjuvants will be well known to those of skill in the art. Exemplary adjuvants include complete or incomplete Freund&#39;s adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). 
     For example, a peptide antigen comprising the novel adaptor/scaffold protein phosphorylation site in SEQ ID NO: 4 shown by the lower case “y” in Table 1 may be used to produce antibodies that specifically bind the novel tyrosine phosphorylation site. 
     When the above-described methods are used for producing polyclonal antibodies, following immunization, the polyclonal antibodies which secreted into the bloodstream can be recovered using known techniques. Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, such as for example, affinity chromatography with Protein A, anti-immunoglobulin, or the antigen itself. In any case, in order to monitor the success of immunization, the antibody levels with respect to the antigen in serum will be monitored using standard techniques such as ELISA, RIA and the like. 
     Monoclonal antibodies of the invention may be produced by any of a number of means that are well-known in the art. In some embodiments, antibody-producing B cells are isolated from an animal immunized with a peptide antigen as described above. The B cells may be from the spleen, lymph nodes or peripheral blood. Individual B cells are isolated and screened as described below to identify cells producing an antibody specific for the novel tyrosine phosphorylation site of interest. Identified cells are then cultured to produce a monoclonal antibody of the invention. 
     Alternatively, a monoclonal phosphorylation site-specific antibody of the invention may be produced using standard hybridoma technology, 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 any of a number of standard means. Methods of immortalizing cells include, but are not limited to, transfecting them with oncogenes, infecting them with an oncogenic virus and cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line). Typically the antibody producing cell and the immortalized cell (such as but not limited to myeloma cells) with which it is fused are from the same species. 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 immortalized antibody producing cells, such as 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. 
     The invention also encompasses antibody-producing cells and cell lines, such as hybridomas, as described above. 
     Polyclonal or monoclonal antibodies may also be obtained through in vitro immunization. For example, phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for a particular antigen. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al., (1994)  EMBO J.,  13:3245-3260; Nissim et al., ibid, pp. 692-698 and by Griffiths et al., ibid, 12:725-734, which are incorporated by reference. 
     The antibodies may be produced recombinantly using methods well known in the art for example, according to the methods disclosed in U.S. Pat. No. 4,349,893 (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.) 
     Once a desired phosphorylation site-specific antibody is identified, polynucleotides encoding the antibody, such as heavy, light chains or both (or single chains in the case of a single chain antibody) or portions thereof such as those encoding the variable region, may be cloned and isolated from antibody-producing cells using means that are well known in the art. 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.) 
     Accordingly, in a further aspect, the invention provides such nucleic acids encoding the heavy chain, the light chain, a variable region, a framework region or a CDR of an antibody of the invention. In some embodiments, the nucleic acids are operably linked to expression control sequences. The invention, thus, also provides vectors and expression control sequences useful for the recombinant expression of an antibody or antigen-binding portion thereof of the invention. Those of skill in the art will be able to choose vectors and expression systems that are suitable for the host cell in which the antibody or antigen-binding portion is to be expressed. 
     Monoclonal antibodies of the invention may be produced recombinantly by expressing the encoding nucleic acids in a suitable host cell under suitable conditions. Accordingly, the invention further provides host cells comprising the nucleic acids and vectors described above. 
     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&#39;l Acad. Sci.  87: 8095 (1990). 
     If monoclonal antibodies of a single desired 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&#39;l. Acad. Sci.,  82: 8653 (1985); Spira et al.,  J. Immunol. Methods,  74: 307 (1984)). Alternatively, the isotype of a monoclonal antibody with desirable propertied can be changed using antibody engineering techniques that are well-known in the art. 
     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 phosphorylated and/or unphosphosphorylated peptide library by ELISA to ensure specificity for both the desired antigen (i.e. that epitope including a phosphorylation site of the invention and for reactivity only with the phosphorylated (or unphosphorylated) form of the antigen. Peptide competition assays may be carried out to confirm lack of reactivity with other phospho-epitopes on the parent protein. The antibodies may also be tested by Western blotting against cell preparations containing the parent signaling protein, e.g., cell lines over-expressing the parent 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 phosphorylation sites with flanking sequences that are highly homologous to that of a phosphorylation site of the invention. 
     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 phosphorylation and activation state and level of a phosphorylation site in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., Antibodies: A Laboratory Manual, Chapter 10, Harlow &amp; 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&#39;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 lysed erythrocytes and cell debris. Adhering cells may be scrapped off plates and washed with PBS. 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 parent signaling 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 may specifically bind to a signaling protein or polypeptide listed in Table 1 only when phosphorylated at the specified tyrosine residue, but are not limited only to binding to the listed signaling proteins of human species, per se. The invention includes antibodies that also bind conserved and highly homologous or identical phosphorylation sites in respective signaling proteins from other species (e.g., mouse, rat, monkey, yeast), in addition to binding the phosphorylation site of the human homologue. The term “homologous” refers to two or more sequences or subsequences that have at least about 85%, at least 90%, at least 95%, or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using sequence comparison method (e.g., BLAST) and/or by visual inspection. Highly homologous or identical sites conserved in other species can readily be identified by standard sequence comparisons (such as BLAST). 
     Methods for making bispecific antibodies are within the purview of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion is with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of illustrative currently known methods for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96/27011; Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. 
     Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. A strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994). Alternatively, the antibodies can be “linear antibodies” as described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. 
     To produce the chimeric antibodies, the portions derived from two different species (e.g., human constant region and murine variable or binding region) can be joined together chemically by conventional techniques or can be prepared as single contiguous proteins using genetic engineering techniques. The DNA molecules encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody can be expressed as contiguous proteins. The method of making chimeric antibodies is disclosed in U.S. Pat. No. 5,677,427; U.S. Pat. No. 6,120,767; and U.S. Pat. No. 6,329,508, each of which is incorporated by reference in its entirety. 
     Fully human antibodies may be produced by a variety of techniques. One example is trioma methodology. The basic approach and an exemplary cell fusion partner, SPAZ-4, for use in this approach have been described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666 (each of which is incorporated by reference in its entirety). 
     Human antibodies can also be produced from non-human transgenic animals having transgenes encoding at least a segment of the human immunoglobulin locus. The production and properties of animals having these properties are described in detail by, see, e.g., Lonberg et al., WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al., WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated by reference in their entirety. 
     Various recombinant antibody library technologies may also be utilized to produce fully human antibodies. For example, one approach is to screen a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047; U.S. Pat. No. 5,969,108, (each of which is incorporated by reference in its entirety). 
     Eukaryotic ribosome can also be used as means to display a library of antibodies and isolate the binding human antibodies by screening against the target antigen, as described in Coia G, et al., J. Immunol. Methods 1: 254 (1-2):191-7 (2001); Hanes J. et al., Nat. Biotechnol. 18(12):1287-92 (2000); Proc. Natl. Acad. Sci. U.S.A. 95(24):14130-5 (1998); Proc. Natl. Acad. Sci. U.S.A. 94(10):4937-42 (1997), each which is incorporated by reference in its entirety. 
     The yeast system is also suitable for screening mammalian cell-surface or secreted proteins, such as antibodies. Antibody libraries may be displayed on the surface of yeast cells for the purpose of obtaining the human antibodies against a target antigen. This approach is described by Yeung, et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E. T., et al., Nat. Biotechnol. 15(6):553-7 (1997), each of which is herein incorporated by reference in its entirety. Alternatively, human antibody libraries may be expressed intracellularly and screened via the yeast two-hybrid system (WO0200729A2, which is incorporated by reference in its entirety). 
     Recombinant DNA techniques can be used to produce the recombinant phosphorylation site-specific antibodies described herein, as well as the chimeric or humanized phosphorylation site-specific antibodies, or any other genetically-altered antibodies and the fragments or conjugate thereof in any expression systems including both prokaryotic and eukaryotic expression systems, such as bacteria, yeast, insect cells, plant cells, mammalian cells (for example, NS0 cells). 
     Once produced, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present application can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, Scopes, R., Protein Purification (Springer-Verlag, N.Y., 1982)). Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent staining, and the like. (See, generally, Immunological Methods, Vols. I and II (Lefkovits and Pernis, eds., Academic Press, NY, 1979 and 1981). 
     6. Therapeutic Uses 
     In a further aspect, the invention provides methods and compositions for therapeutic uses of the peptides or proteins comprising a phosphorylation site of the invention, and phosphorylation site-specific antibodies of the invention. 
     In one embodiment, the invention provides for a method of treating or preventing carcinoma in a subject, wherein the carcinoma is associated with the phosphorylation state of a novel phosphorylation site in Table 1, whether phosphorylated or dephosphorylated, comprising: administering to a subject in need thereof a therapeutically effective amount of a peptide comprising a novel phosphorylation site (Table 1) and/or an antibody or antigen-binding fragment thereof that specifically bind a novel phosphorylation site of the invention (Table 1). The antibodies maybe full-length antibodies, genetically engineered antibodies, antibody fragments, and antibody conjugates of the invention. 
     The term “subject” refers to a vertebrate, such as for example, a mammal, or a human. Although present application are primarily concerned with the treatment of human subjects, the disclosed methods may also be used for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes. 
     In one aspect, the disclosure provides a method of treating carcinoma in which a peptide or an antibody that reduces at least one biological activity of a targeted signaling protein is administered to a subject. For example, the peptide or the antibody administered may disrupt or modulate the interaction of the target signaling protein with its ligand. Alternatively, the peptide or the antibody may interfere with, thereby reducing, the down-stream signal transduction of the parent signaling protein. An antibody that specifically binds the novel tyrosine phosphorylation site only when the tyrosine is phosphorylated, and that does not substantially bind to the same sequence when the tyrosine is not phosphorylated, thereby prevents downstream signal transduction triggered by a phospho-tyrosine. Alternatively, an antibody that specifically binds the unphosphorylated target phosphorylation site reduces the phosphorylation at that site and thus reduces activation of the protein mediated by phosphorylation of that site. Similarly, an unphosphorylated peptide may compete with an endogenous phosphorylation site for same kinases, thereby preventing or reducing the phosphorylation of the endogenous target protein. Alternatively, a peptide comprising a phosphorylated novel tyrosine site of the invention but lacking the ability to trigger signal transduction may competitively inhibit interaction of the endogenous protein with the same down-stream ligand(s). 
     The antibodies of the invention may also be used to target cancer cells for effector-mediated cell death. The antibody disclosed herein may be administered as a fusion molecule that includes a phosphorylation site-targeting portion joined to a cytotoxic moiety to directly kill cancer cells. Alternatively, the antibody may directly kill the cancer cells through complement-mediated or antibody-dependent cellular cytotoxicity. 
     Accordingly in one embodiment, the antibodies of the present disclosure may be used to deliver a variety of cytotoxic compounds. Any cytotoxic compound can be fused to the present antibodies. The fusion can be achieved chemically or genetically (e.g., via expression as a single, fused molecule). The cytotoxic compound can be a biological, such as a polypeptide, or a small molecule. As those skilled in the art will appreciate, for small molecules, chemical fusion is used, while for biological compounds, either chemical or genetic fusion can be used. 
     Non-limiting examples of cytotoxic compounds include therapeutic drugs, radiotherapeutic agents, ribosome-inactivating proteins (RIPs), chemotherapeutic agents, toxic peptides, toxic proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short-range radiation emitters, including, for example, short-range, high-energy α-emitters. Enzymatically active toxins and fragments thereof, including ribosome-inactivating proteins, are exemplified by saporin, luffin, momordins, ricin, trichosanthin, gelonin, abrin, etc. Procedures for preparing enzymatically active polypeptides of the immunotoxins are described in WO84/03508 and WO85/03508, which are hereby incorporated by reference. Certain cytotoxic moieties are derived from adriamycin, chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum, for example. 
     Exemplary chemotherapeutic agents that may be attached to an antibody or antigen-binding fragment thereof include taxol, doxorubicin, verapamil, podophyllotoxin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, transplatinum, 5-fluorouracil, vincristin, vinblastin, or methotrexate. 
     Procedures for conjugating the antibodies with the cytotoxic agents have been previously described and are within the purview of one skilled in the art. 
     Alternatively, the antibody can be coupled to high energy radiation emitters, for example, a radioisotope, such as  131 I, a γ-emitter, which, when localized at the tumor site, results in a killing of several cell diameters. See, e.g., S. E. Order, “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-316 (Academic Press 1985), which is hereby incorporated by reference. Other suitable radioisotopes include α-emitters, such as  212 Bi,  213 Bi, and  211 At, and β-emitters, such as  186 Re and  90 Y. 
     Because many of the signaling proteins in which novel tyrosine phosphorylation sites of the invention occur also are expressed in normal cells and tissues, it may also be advantageous to administer a phosphorylation site-specific antibody with a constant region modified to reduce or eliminate ADCC or CDC to limit damage to normal cells. For example, effector function of an antibodies may be reduced or eliminated by utilizing an IgG1 constant domain instead of an IgG2/4 fusion domain. Other ways of eliminating effector function can be envisioned such as, e.g., mutation of the sites known to interact with FcR or insertion of a peptide in the hinge region, thereby eliminating critical sites required for FcR interaction. Variant antibodies with reduced or no effector function also include variants as described previously herein. 
     The peptides and antibodies of the invention may be used in combination with other therapies or with other agents. Other agents include but are not limited to polypeptides, small molecules, chemicals, metals, organometallic compounds, inorganic compounds, nucleic acid molecules, oligonucleotides, aptamers, spiegelmers, antisense nucleic acids, locked nucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, immunomodulatory agents, antigen-binding fragments, prodrugs, and peptidomimetic compounds. In certain embodiments, the antibodies and peptides of the invention may be used in combination with cancer therapies known to one of skill in the art. 
     In certain aspects, the present disclosure relates to combination treatments comprising a phosphorylation site-specific antibody described herein and immunomodulatory compounds, vaccines or chemotherapy. Illustrative examples of suitable immunomodulatory agents that may be used in such combination therapies include agents that block negative regulation of T cells or antigen presenting cells (e.g., anti-CTLA4 antibodies, anti-PD-L1 antibodies, anti-PDL-2 antibodies, anti-PD-1 antibodies and the like) or agents that enhance positive co-stimulation of T cells (e.g., anti-CD40 antibodies or anti 4-1BB antibodies) or agents that increase NK cell number or T-cell activity (e.g., inhibitors such as IMiDs, thalidomide, or thalidomide analogs). Furthermore, immunomodulatory therapy could include cancer vaccines such as dendritic cells loaded with tumor cells, proteins, peptides, RNA, or DNA derived from such cells, patient derived heat-shock proteins (hsp&#39;s) or general adjuvants stimulating the immune system at various levels such as CpG, Luivac®, Biostim®, Ribomunyl®, Imudon®, Bronchovaxom® or any other compound or other adjuvant activating receptors of the innate immune system (e.g., toll like receptor agonist, anti-CTLA-4 antibodies, etc.). Also, immunomodulatory therapy could include treatment with cytokines such as IL-2, GM-CSF and IFN-gamma. 
     Furthermore, combination of antibody therapy with chemotherapeutics could be particularly useful to reduce overall tumor burden, to limit angiogenesis, to enhance tumor accessibility, to enhance susceptibility to ADCC, to result in increased immune function by providing more tumor antigen, or to increase the expression of the T cell attractant LIGHT. 
     Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. 
     These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into groups, including, for example, the following classes of agents: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate inhibitors and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); immunomodulatory agents (thalidomide and analogs thereof such as lenalidomide (Revlimid, CC-5013) and CC-4047 (Actimid)), cyclophosphamide; anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. 
     In certain embodiments, pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of “angiogenic molecules,” such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as anti-βbFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D 3  analogs, alpha-interferon, and the like. For additional proposed inhibitors of angiogenesis, see Blood et al., Biochim. Biophys. Acta, 1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6,573,256. In addition, there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF-mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), troponin subunits, inhibitors of vitronectin α v β 3 , peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM-138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845. 
     7. Diagnostic Uses 
     In a further aspect, the invention provides methods for detecting and quantitating phosphoyrlation at a novel tyrosine phosphorylation site of the invention. For example, peptides, including AQUA peptides of the invention, and antibodies of the invention are useful in diagnostic and prognostic evaluation of carcinomas, wherein the carcinoma is associated with the phosphorylation state of a novel phosphorylation site in Table 1, whether phosphorylated or dephosphorylated. 
     Methods of diagnosis can be performed in vitro using a biological sample (e.g., blood sample, lymph node biopsy or tissue) from a subject, or in vivo. The phosphorylation state or level at the tyrosine residue identified in the corresponding row in Column D of Table 1 may be assessed. A change in the phosphorylation state or level at the phosphorylation site, as compared to a control, indicates that the subject is suffering from, or susceptible to, carcinoma. 
     In one embodiment, the phosphorylation state or level at a novel phosphorylation site is determined by an AQUA peptide comprising the phosphorylation site. The AQUA peptide may be phosphorylated or unphosphorylated at the specified tyrosine position. 
     In another embodiment, the phosphorylation state or level at a phosphorylation site is determined by an antibody or antigen-binding fragment thereof, wherein the antibody specifically binds the phosphorylation site. The antibody may be one that only binds to the phosphorylation site when the tyrosine residue is phosphorylated, but does not bind to the same sequence when the tyrosine is not phosphorylated; or vice versa. 
     In particular embodiments, the antibodies of the present application are attached to labeling moieties, such as a detectable marker. One or more detectable labels can be attached to the antibodies. Exemplary labeling moieties include radiopaque dyes, radiocontrast agents, fluorescent molecules, spin-labeled molecules, enzymes, or other labeling moieties of diagnostic value, particularly in radiologic or magnetic resonance imaging techniques. 
     A radiolabeled antibody in accordance with this disclosure can be used for in vitro diagnostic tests. The specific activity of an antibody, binding portion thereof, probe, or ligand, depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the biological agent. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity. Radioisotopes useful as labels, e.g., for use in diagnostics, include iodine ( 131 I or  125 I), indium ( 111 In), technetium ( 99 Tc), phosphorus ( 32 P), carbon ( 14 C), and tritium ( 3 H), or one of the therapeutic isotopes listed above. 
     Fluorophore and chromophore labeled biological agents can be prepared from standard moieties known in the art. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties may be selected to have substantial absorption at wavelengths above 310 nm, such as for example, above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer, Science, 162:526 (1968) and Brand et al., Annual Review of Biochemistry, 41:843-868 (1972), which are hereby incorporated by reference. The antibodies can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated by reference. 
     The control may be parallel samples providing a basis for comparison, for example, biological samples drawn from a healthy subject, or biological samples drawn from healthy tissues of the same subject. Alternatively, the control may be a pre-determined reference or threshold amount. If the subject is being treated with a therapeutic agent, and the progress of the treatment is monitored by detecting the tyrosine phosphorylation state level at a phosphorylation site of the invention, a control may be derived from biological samples drawn from the subject prior to, or during the course of the treatment. 
     In certain embodiments, antibody conjugates for diagnostic use in the present application are intended for use in vitro, where the antibody is linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. In certain embodiments, secondary binding ligands are biotin and avidin or streptavidin compounds. 
     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 signaling protein in subjects before, during, and after treatment with a therapeutic agent targeted at inhibiting tyrosine phosphorylation 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 signaling 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). 
     Alternatively, antibodies of the invention may be used 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. 
     Peptides and 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 the phosphorylation state or level at two or more phosphorylation sites of the invention (Table 1) in a biological sample, the method comprising utilizing two or more antibodies or AQUA peptides of the invention. In one preferred embodiment, two to five antibodies or AQUA peptides of the invention are used. In another preferred embodiment, six to ten antibodies or AQUA peptides of the invention are used, while in another preferred embodiment eleven to twenty antibodies or AQUA peptides of the invention are used. 
     In certain embodiments the diagnostic methods of the application may be used in combination with other cancer diagnostic tests. 
     The biological sample analyzed may be any sample that is suspected of having abnormal tyrosine phosphorylation at a novel phosphorylation site of the invention, such as a homogenized neoplastic tissue sample. 
     8. Screening assays 
     In another aspect, the invention provides a method for identifying an agent that modulates tyrosine phosphorylation at a novel phosphorylation site of the invention, comprising: a) contacting a candidate agent with a peptide or protein comprising a novel phosphorylation site of the invention; and b) determining the phosphorylation state or level at the novel phosphorylation site. A change in the phosphorylation level of the specified tyrosine in the presence of the test agent, as compared to a control, indicates that the candidate agent potentially modulates tyrosine phosphorylation at a novel phosphorylation site of the invention. 
     In one embodiment, the phosphorylation state or level at a novel phosphorylation site is determined by an AQUA peptide comprising the phosphorylation site. The AQUA peptide may be phosphorylated or unphosphorylated at the specified tyrosine position. 
     In another embodiment, the phosphorylation state or level at a phosphorylation site is determined by an antibody or antigen-binding fragment thereof, wherein the antibody specifically binds the phosphorylation site. The antibody may be one that only binds to the phosphorylation site when the tyrosine residue is phosphorylated, but does not bind to the same sequence when the tyrosine is not phosphorylated; or vice versa. 
     In particular embodiments, the antibodies of the present application are attached to labeling moieties, such as a detectable marker. 
     The control may be parallel samples providing a basis for comparison, for example, the phosphorylation level of the target protein or peptide in absence of the testing agent. Alternatively, the control may be a pre-determined reference or threshold amount. 
     9. Immunoassays 
     In another aspect, the present application concerns immunoassays for binding, purifying, quantifying and otherwise generally detecting the phosphorylation state or level at a novel phosphorylation site of the invention. 
     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 used 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 using 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. 
     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. 
     In certain embodiments, immunoassays are the various types of enzyme linked immunoadsorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot and slot blotting, FACS analyses, and the like may also be used. The steps of various useful immunoassays have been described in the scientific literature, such as, e.g., Nakamura et al., in Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Chapter 27 (1987), incorporated herein by reference. 
     In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are based upon the detection of radioactive, fluorescent, biological or enzymatic tags. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art. 
     The antibody used in the detection may itself be conjugated to a detectable label, wherein one would then simply detect this label. The amount of the primary immune complexes in the composition would, thereby, be determined. 
     Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are washed extensively to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is detected. 
     An enzyme linked immunoadsorbent assay (ELISA) is a type of binding assay. In one type of ELISA, phosphorylation site-specific antibodies disclosed herein are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a suspected neoplastic tissue sample is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound target signaling protein may be detected. 
     In another type of ELISA, the neoplastic tissue samples are immobilized onto the well surface and then contacted with the phosphorylation site-specific antibodies disclosed herein. After binding and washing to remove non-specifically bound immune complexes, the bound phosphorylation site-specific antibodies are detected. 
     Irrespective of the format used, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. 
     The radioimmunoassay (RIA) is an analytical technique which depends on the competition (affinity) of an antigen for antigen-binding sites on antibody molecules. Standard curves are constructed from data gathered from a series of samples each containing the same known concentration of labeled antigen, and various, but known, concentrations of unlabeled antigen. Antigens are labeled with a radioactive isotope tracer. The mixture is incubated in contact with an antibody. Then the free antigen is separated from the antibody and the antigen bound thereto. Then, by use of a suitable detector, such as a gamma or beta radiation detector, the percent of either the bound or free labeled antigen or both is determined. This procedure is repeated for a number of samples containing various known concentrations of unlabeled antigens and the results are plotted as a standard graph. The percent of bound tracer antigens is plotted as a function of the antigen concentration. Typically, as the total antigen concentration increases the relative amount of the tracer antigen bound to the antibody decreases. After the standard graph is prepared, it is thereafter used to determine the concentration of antigen in samples undergoing analysis. 
     In an analysis, the sample in which the concentration of antigen is to be determined is mixed with a known amount of tracer antigen. Tracer antigen is the same antigen known to be in the sample but which has been labeled with a suitable radioactive isotope. The sample with tracer is then incubated in contact with the antibody. Then it can be counted in a suitable detector which counts the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent may also be similarly counted. Then, from the standard curve, the concentration of antigen in the original sample is determined. 
     10. Pharmaceutical Formulations and Methods of Administration 
     Methods of administration of therapeutic agents, particularly peptide and antibody therapeutics, are well-known to those of skill in the art. 
     Peptides of the invention can be administered in the same manner as conventional peptide type pharmaceuticals. Preferably, peptides are administered parenterally, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously. When administered orally, peptides may be proteolytically hydrolyzed. Therefore, oral application may not be usually effective. However, peptides can be administered orally as a formulation wherein peptides are not easily hydrolyzed in a digestive tract, such as liposome-microcapsules. Peptides may be also administered in suppositories, sublingual tablets, or intranasal spray. 
     If administered parenterally, a preferred pharmaceutical composition is an aqueous solution that, in addition to a peptide of the invention as an active ingredient, may contain for example, buffers such as phosphate, acetate, etc., osmotic pressure-adjusting agents such as sodium chloride, sucrose, and sorbitol, etc., antioxidative or antioxygenic agents, such as ascorbic acid or tocopherol and preservatives, such as antibiotics. The parenterally administered composition also may be a solution readily usable or in a lyophilized form which is dissolved in sterile water before administration. 
     The pharmaceutical formulations, dosage forms, and uses described below generally apply to antibody-based therapeutic agents, but are also useful and can be modified, where necessary, for making and using therapeutic agents of the disclosure that are not antibodies. 
     To achieve the desired therapeutic effect, the phosphorylation site-specific antibodies or antigen-binding fragments thereof can be administered in a variety of unit dosage forms. The dose will vary according to the particular antibody. For example, different antibodies may have different masses and/or affinities, and thus require different dosage levels. Antibodies prepared as Fab or other fragments will also require differing dosages than the equivalent intact immunoglobulins, as they are of considerably smaller mass than intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient&#39;s blood. The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. Dosage levels of the antibodies for human subjects are generally between about 1 mg per kg and about 100 mg per kg per patient per treatment, such as for example, between about 5 mg per kg and about 50 mg per kg per patient per treatment. In terms of plasma concentrations, the antibody concentrations may be in the range from about 25 μg/mL to about 500 μg/mL. However, greater amounts may be required for extreme cases and smaller amounts may be sufficient for milder cases. 
     Administration of an antibody will generally be performed by a parenteral route, typically via injection such as intra-articular or intravascular injection (e.g., intravenous infusion) or intramuscular injection. Other routes of administration, e.g., oral (p.o.), may be used if desired and practicable for the particular antibody to be administered. An antibody can also be administered in a variety of unit dosage forms and their dosages will also vary with the size, potency, and in vivo half-life of the particular antibody being administered. Doses of a phosphorylation site-specific antibody will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. 
     The frequency of administration may also be adjusted according to various parameters. These include the clinical response, the plasma half-life of the antibody, and the levels of the antibody in a body fluid, such as, blood, plasma, serum, or synovial fluid. To guide adjustment of the frequency of administration, levels of the antibody in the body fluid may be monitored during the course of treatment. 
     Formulations particularly useful for antibody-based therapeutic agents are also described in U.S. Patent App. Publication Nos. 20030202972, 20040091490 and 20050158316. In certain embodiments, the liquid formulations of the application are substantially free of surfactant and/or inorganic salts. In another specific embodiment, the liquid formulations have a pH ranging from about 5.0 to about 7.0. In yet another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from about 1 mM to about 100 mM. In still another specific embodiment, the liquid formulations comprise histidine at a concentration ranging from 1 mM to 100 mM. It is also contemplated that the liquid formulations may further comprise one or more excipients such as a saccharide, an amino acid (e.g., arginine, lysine, and methionine) and a polyol. Additional descriptions and methods of preparing and analyzing liquid formulations can be found, for example, in PCT publications WO 03/106644, WO 04/066957, and WO 04/091658. 
     Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the application. 
     In certain embodiments, formulations of the subject antibodies are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside microorganisms and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food &amp; Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin. 
     The amount of the formulation which will be therapeutically effective can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient&#39;s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient&#39;s symptoms. For example, the actual patient body weight may be used to calculate the dose of the formulations in milliliters (mL) to be administered. There may be no downward adjustment to “ideal” weight. In such a situation, an appropriate dose may be calculated by the following formula: 
       Dose (mL)=[patient weight (kg)×dose level (mg/kg)/drug concentration (mg/mL)] 
     For the purpose of treatment of disease, the appropriate dosage of the compounds (for example, antibodies) will depend on the severity and course of disease, the patient&#39;s clinical history and response, the toxicity of the antibodies, and the discretion of the attending physician. The initial candidate dosage may be administered to a patient. The proper dosage and treatment regimen can be established by monitoring the progress of therapy using conventional techniques known to those of skill in the art. 
     The formulations of the application can be distributed as articles of manufacture comprising packaging material and a pharmaceutical agent which comprises, e.g., the antibody and a pharmaceutically acceptable carrier as appropriate to the mode of administration. The packaging material will include a label which indicates that the formulation is for use in the treatment of prostate cancer. 
     11. Kits 
     Antibodies and peptides (including AQUA peptides) of the invention may also be used within a kit for detecting the phosphorylation state or level at a novel phosphorylation site of the invention, comprising at least one of the following: an AQUA peptide comprising the phosphorylation site, or an antibody or an antigen-binding fragment thereof that binds to an amino acid sequence comprising the phosphorylation site. Such a kit may further comprise a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and co-factors required by the enzyme. In addition, other additives may be included such as stabilizers, buffers and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that, on dissolution, will provide a reagent solution having the appropriate concentration. 
     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 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 novel tyrosine phosphorylation sites in leukemia, IAP isolation techniques were used to identify phosphotyrosine-containing peptides in cell extracts from human leukemia cell lines and patient cell lines identified in Column G of Table 1 including: 23132/87; 293T; 293T(ATIC-ALK); 293T(NPM-ALK); 293T(NPM-ALKIATIC-ALK); 293T(ZNF198-FGFR); 3T3; 3T3(Abl); 3T3(Src); 42-MG-BA; 5637; 639L; 8-MG-BA; A 431; A 431: EGF, Iressa; A172; A498; A549; A704; AGS; ARH-77; AU-565; B13_AML; B16_AML; B17_AML; B18_AML; B20-XY1; B23-XY2; B24-XY2; B24_AML; B25-XY2; B25_AML; B29-XY2; B29_AML; B30-XY2; B32-XY2; B34-XY2; B36-XY2; B37-XY2; B38-XY2; B39-XY2; B41-XY2; BC-3C; BC001; BC002; BC003; BC004; BC005; BC007; BC008; BJ629; BJ630; BJ631; BJ635; BJ665; BJ669; BT-20; BT-549; BT1; BT2; C2C12-D; C2C12-D: Insulin; CAKI-2; CAL-29; CAL-51; CAL-85-1; CAMA-1; CAS-1; CCF-STTG1; CHP-212; CHP126; CHRF; CI-1; CMK; CMS; COLO-699; CTV-1; CTV-1: PP2; Cal-12T; Cal-148; Calu-3; CaoV3; Colo-704; Colo-824; Colo680N; DBTRG-05MG; DK-MG; DMS153; DMS 53; DMS 79; DND-41; DU-4475; DU.528; DU145; DV-90; Detroit562; EFM-19; EFM-192A; EFO-21; EFO-27; ELF-153; ENT01; ENT02; ENT03; ENT04; ENT05; ENT10; ENT12; ENT14; ENT15; ENT16; ENT17; ENT18; ENT19; ENT20; ENT23; ENT25; ENT26; ENT7; ENT8; ENT9; EOL-1; ES2; EVSA-T; FUOV1; GAMG; GI-CA-N; GMS-10; H128; H1299; H1355; H1373; H1417; H1435; H1437; H1563; H1648; H1650; H1666; H1693; H1703; H1734; H1781; H1793; H1838; H1869; H1915; H1944; H1975; H1993; H2023; H2030; H2052; H2066; H2085; H209; H2135; H2170; H2172; H2286; H2342; H2347; H2452; H28; H3255; H358; H4; H441; H446; H460; H520; H524; H526; H596; H647; H661; H810; H82; H838; H929; HCC1143; HCC1187; HCC1395; HCC1419; HCC1428; HCC15; HCC1500; HCC1569; HCC1599; HCC1806; HCC1937; HCC202; HCC366; HCC38; HCC44; HCC70; HCC78; HCC78: TSA 24h; HCC827; HCC827: Geldanamycin; HCC827: TSA; HCT116; HCT15; HCT15: TSA; HCT8; HCT8: TSA; HD-MyZ; HDLM-2; HDQ-P1; HEL; HEL: Flt3 inhibitor; HEL: Jak Inhibitor I; HL107A; HL107B; HL116A; HL116B; HL117A; HL117B; HL127A; HL127B; HL129A; HL130A; HL131A; HL131B; HL132A; HL132B; HL133A; HL137A; HL144A; HL144B; HL145A; HL145B; HL146A; HL146B; HL148A; HL148B; HL150A; HL150B; HL151A; HL151B; HL152B; HL183A; HL183B; HL184A; HL184B; HL213A; HL226A; HL226B; HL233A; HL233B; HL234A; HL234B; HL235A; HL25A; HL53A; HL53B; HL55A; HL55B; HL57; HL59A; HL59B; HL61A; HL61B; HL61b; HL66A; HL66B; HL68A; HL75A; HL76A; HL76B; HL79A; HL79B; HL83A; HL84A; HL84B; HL87A; HL87B; HL92A; HL92B; HL94A; HL94B; HL97A; HL97B; HL98A; HP28; HPAC: EGF; HT29; HU-3; Hs.683; Hs746T; Hs766T; IMR32; J82; JIMT-1; June07cs148; June07cs161; June07cs180; Jurkat: anti-CD3, anti-mouse Ig, anti-CD28; Jurkat: calyculin, pervanadate; Jurkat: pervanadate; Jurkat: pervanadate, calyculin; K562; KATO III; KBM-3; KELLY; KG-1; KMS-27; KOPT-K1; KPL-1; KY821; Karpas 299; Karpas-1106P; Kyse140; Kyse180; Kyse270; Kyse30; Kyse410; Kyse450; Kyse510; Kyse520; Kyse70; L428; L540; LAN-1; LAN-5; LCLC-103H; LN-405; LN18; LN229; LNCaP; LOU-NH91; LP-1; LXF-289; M-07e; M059J; M059K; MC-116; MCF7; MDA-MB-134vi; MDA-MB-157; MDA-MB-175vii; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; MDA-MB-468: EGF; MDAH2774; MEC-2; MHH-CALL4; MHH-NB-11; MIAPaCa-2; MIAPaCa-2: EGF; MKN-45; MKPL-1; ML-1; MNNG/MOS; MONO-MAC-6; MT-3; MUTZ-5; MV4-11; MV4-11: DMSO; MV4-11: SAHA 3h; MV4-11: SAHA 6h; MV4-11∥pervanadate; Marimo; Me-F2; Molm 14; Molt 15; N06211(1); N06218(1); N06218(2); N06BJ504(1); N06BJ504-R; N06BJ505(2); N06BJ526(21); N06BJ530(6); N06BJ573(9); N06BJ591(11); N06BJ593(13); N06BJ601(18); N06BJ606(19); N06C45AG-R; N06CS02; N06CS06; N06CS09; N06CS106; N06CS107; N06CS110AG-R; N06CS113-R; N06CS16; N06CS17; N06CS22(2)-R; N06CS22-1; N06CS22-2; N06CS23; N06CS34; N06CS38; N06CS39; N06CS40; N06CS75; N06CS77; N06CS82; N06CS83; N06CS87; N06CS89; N06CS90; N06CS91; N06CS93-1; N06CS93-2; N06CS94; N06CS97; N06CS97-R; N06CS98; N06CS98-2; N06N101; N06N 102 ; N06N103; N06N 106 ; N06N109; N06N121; N06N127; N06N128; N06N129; N06N130; N06N131; N06N132; N06N75; N06N80; N06N90; N06N93; N06bj523(3); N06bj567(7); N06bj570(8); N06bj590(10); N06bj592(12); N06bj594(14); N06bj595(15); N06bj596(16); N06bj598(17); N06bj632(24); N06bj638(26); N06bj639(27); N06bj667(29); N06c144; N06c78; N06cs109; N06cs110; N06cs110-R; N06cs111; N06cs112; N06cs113; N06cs115; N06cs117; N06cs121; N06cs122; N06cs122-R; N06cs123; N06cs123(2); N06cs126; N06cs128; N06cs129; N06cs130; N06cs132; N06cs133; N06cs21; N06cs49; N06cs59; N06cs63; N06cs88; N06cs92; N06cs95; N87; N87: EGF; NALM-19; NCI-H716; NKM-1; Nomo-1; Nomo-1: DMSO; Nomo-1: SAHA 3h; Nomo-1: SAHA 6h; OCI-M1; OCI/AML2; OCI/AML3; OV90; PA-1; PL21; PL21∥pervanadate; Pfeiffer; RC-K8; RI-1; RKO: mutBRaf; RPMI-8266; RS4; SEM; SH-SY5Y; SIMA; SK-BR-3; SK-N-AS; SK-N-BE(2); SK-N-DZ; SK-N-FI; SK-N-MC; SK-N-SH; SK-OV-3; SNB-19; SNU-1; SNU-16; SNU-5; SNU-C2B; SNU-C2B: TSA; SU-DHL1; SU-DHL4; SUP-T13; SW1088; SW1710; SW1783; SW480; SW620; SW620: TSA; SW780; SW780; Scaber; SuDHL5; SuDHL8; T17; T98G; Thom; Thom(MPL, W515L); U118 MG; UACC-812; UACC-893; UM-UC-1; UT-7; VAC0432: mutBRaf; VAL; WSU-NHL; ZR-75-1; ZR-75-30; brain; brain: ischemia; cs001; cs012; cs015; cs018; cs019; cs024; cs025; cs026; cs029; cs037; cs041; cs042; cs048; cs057; cs068; cs069; cs070; cs103; cs104; cs105; cs106; cs107; cs110; cs111; cs114; cs131; cs133; cs136; cs153; csBC001; csC43; csC44; csC45; csC50; csC52; csC56; csC58; csC60; csC62; csC66; csC71; gz21; gz30; gz33; gz42; gz47; gz52; gz56; gz58; gz61; gz62; gz63; gz68; gz7; gz70; gz73; gz74; gz75; gzB1; h2073; h2228; lung (mouse); mouse heart; mouse liver; sw48; and sw48: TSA. 
     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 1.25×10 8  cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyro-phosphate, 1 mM β-glycerol-phosphate) and sonicated. 
     Adherent cells at about 70-80% 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 2×10 8  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. 
     Frozen tissue samples were cut to small pieces, homogenize in lysis buffer (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, 1 ml lysis buffer for 100 mg of frozen tissue) using a polytron for 2 times of 20 sec. each time. Homogenate is then briefly 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 day 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 C 18  columns (Waters) equilibrated with 0.1% TFA. A column volume of 0.7-1.0 ml was used per 2×10 8  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 2×10 8  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 1.4 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 C18 microtips (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 &amp; 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, 40; minimum TIC, 2×10 3 ; 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, 1.0; 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 then current NCBI human protein database. 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 phosphopeptide sequence is assigned to co-eluting ions with different charge states, since the MS/MS spectrum changes markedly with charge state; (ii) the phosphorylation 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 phosphorylation site is found in more than one peptide sequence context due to homologous but not identical protein isoforms; (iv) the phosphorylation site is found in more than one peptide sequence context due to homologous but not identical proteins among species; and (v) phosphorylation 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 used 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&lt;6, XCorr≧2.2, and DeltaCN&gt;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 Phosphorylation site-Specific Polyclonal Antibodies 
     Polyclonal antibodies that specifically bind a novel phosphorylation site of the invention (Table 1/ FIG. 2 ) only when the tyrosine residue is phosphorylated (and does not bind to the same sequence when the tyrosine is not phosphorylated), and vice versa, are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site and then immunizing an animal to raise antibodies against the antigen, as further described below. Production of exemplary polyclonal antibodies is provided below. 
     A. DLL1 (Tyrosine 641). 
     A 13 amino acid phospho-peptide antigen, YPAVDy*NLVQDLK (SEQ NO: 17; y*=phosphotyrosine), which comprises the phosphorylation site derived from human DLL1 (an adhesion of extracellular matrix protein, Tyr 641 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phosphorylation site-specific polyclonal antibodies as described in Immunization/Screening below. 
     B. Radixin (Tyrosine 270). 
     An 10 amino acid phospho-peptide antigen, APDFVFy*APR (SEQ ID NO: 78; y*=phosphotyrosine), which comprises the phosphorylation site derived from human radixin (a cytoskeletal protein, Tyr 270 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phosphorylation site-specific polyclonal antibodies as described in Immunization/Screening below. 
     C. AKR1C1 (Tyrosine 55). 
     A 19 amino acid phospho-peptide antigen, HIDSAHLy*NNEEQVGLAIR (SEQ ID NO: 86; y*=phosphotyrosine, which comprises the phosphorylation site derived from human AKR1C1 (an enzyme protein, Tyr 55 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phosphorylation site-specific polyclonal 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 A NTIBODIES : A L ABORATORY  M ANUAL , Cold Spring Harbor, supra.). The eluted immunoglobulins are further loaded onto an unphosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the unphosphorylated form of the phosphorylation sites. 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 phosphorylation sites. After washing the column extensively, the bound antibodies (i.e. antibodies that bind the phosphorylated peptides described in A-C above, but do not bind the unphosphorylated form of the peptides) 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 DLL1, radixin or AKR1C1), for example, MDA-MB-453, gz47 or DU145. 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 μl 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 C ELL  S IGNALING  T ECHNOLOGY , I NC . 2003-04 Catalogue, p. 390. The isolated phosphorylation site-specific antibody is used at dilution 1:1000. Phospho-specificity of the antibody will be shown by binding of only the phosphorylated form of the target amino acid sequence. Isolated phosphorylation site-specific polyclonal antibody does not (substantially) recognize the same target sequence when not phosphorylated at the specified tyrosine position (e.g., the antibody does not bind to AKR1C1 in the non-stimulated cells, when tyrosine 55 is not phosphorylated). 
     In order to confirm the specificity of the isolated antibody, different cell lysates containing various phosphorylated signaling proteins other than the target protein are prepared. The Western blot assay is performed again using these cell lysates. The phosphorylation site-specific polyclonal antibody isolated as described above is used (1:1000 dilution) to test reactivity with the different phosphorylated non-target proteins. The phosphorylation site-specific antibody does not significantly cross-react with other phosphorylated signaling proteins that do not have the described phosphorylation site, although occasionally slight binding to a highly homologous sequence 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 Phosphorylation site-specific Monoclonal Antibodies 
     Monoclonal antibodies that specifically bind a novel phosphorylation site of the invention (Table 1) only when the tyrosine residue is phosphorylated (and does not bind to the same sequence when the tyrosine is not phosphorylated) are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site 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. KIF5B (Tyrosine 649). 
     A 12 amino acid phospho-peptide antigen, SLTEy*LQNVEQK (SEQ ID NO: 131; y*=phosphotyrosine), which comprises the phosphorylation site derived from human KIF5B (a motor or contractile protein, Tyr 649 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , 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 phosphorylation site-specific monoclonal antibodies as described in Immunization/Fusion/Screening below. 
     B. Fyn (Tyrosine 185). 
     A 14 amino acid phospho-peptide antigen, ESETTKGAy*SLSIR (SEQ ID NO: 168; y*=phosphotyrosine), which comprises the phosphorylation site derived from human Fyn (a protein kinase, Tyr 185 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , 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 phosphorylation site-specific monoclonal antibodies as described in Immunization/Fusion/Screening below. 
     C. Yes (Tyrosine 222). 
     A 12 amino acid phospho-peptide antigen, KLDNGGy*YITTR (SEQ ID NO: 172; y*=phosphotyrosines), which comprises the phosphorylation site derived from human Yes (a cell cycle regulation protein, Tyr 222 being the phosphorylatable residue), 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 A NTIBODIES : A L ABORATORY  M ANUAL , 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 phosphorylation site-specific monoclonal 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 KIF5B, Fyn and Yes) 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. 
     Example 4 
     Production and Use of AQUA Peptides for Detecting and Quantitating Phosphorylation at a Novel Phosphorylation Site 
     Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detecting and quantitating a novel phosphorylation site of the invention (Table 1) only when the tyrosine residue is phosphorylated 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 MS″ 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. EphA5 (Tyrosine 833). 
     An AQUA peptide comprising the sequence, VLEDDPEAAy*TTR (SEQ ID NO: 176; y*=phosphotyrosine; Valine being  14 C/ 15 N-labeled, as indicated in bold), which comprises the phosphorylation site derived from EphA5 (a protein kinase, Tyr 833 being the phosphorylatable residue), 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 &amp; MS/MS Signature. The EphA5 (tyr 833) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated EphA5 (tyr 833) in the sample, as further described below in Analysis &amp; Quantification. 
     B. ATP6V0A1 (tyrosine 364). 
     An AQUA peptide comprising the sequence MQNQTPPTy*NKTNK (SEQ ID NO: 183 y*=phosphotyrosine; Proline being  14 C/ 15 N-labeled, as indicated in bold), which comprises the phosphorylation site derived from human ATP6V0A1 (Tyr 364) being the phosphorylatable residue), 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 &amp; MS/MS Signature. The ATP6V0A1 (Tyr 364) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated ATP6V0A1 (Tyr 364) in the sample, as further described below in Analysis &amp; Quantification. 
     C. GABRB2 (Tyrosine 98). 
     An AQUA peptide comprising the sequence DKRLSy*NVIPLNLTLDNR (SEQ ID NO: 192; y*=phosphotyrosine; Leucine being  14 C/ 15 N-labeled, as indicated in bold), which comprises the phosphorylation site derived from human GABRB2 (Tyr 98 being the phosphorylatable residue), 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 &amp; MS/MS Signature. The GABRB2 (Tyr 98) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated GABRB2 (Tyr 98) in the sample, as further described below in Analysis &amp; Quantification. 
     D. MLLT11 (Tyrosine 9). 
     An AQUA peptide comprising the sequence DPVSSQySSFLFWR (SEQ ID NO: 314; y*=phosphotyrosine; proline being  14 C/ 15 N-labeled, as indicated in bold), which comprises the phosphorylation site derived from human MLLT11 (Tyr 9 being the phosphorylatable residue), 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 &amp; MS/MS Signature. The MLLT11 (Tyr 9) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated MLLT11 (Tyr 9) in the sample, as further described below in Analysis &amp; Quantification. 
     Synthesis &amp; MS/MS Spectra. 
     Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, Calif.). Fmoc-derivatized stable-isotope monomers containing one  15 N and five to nine  13 C 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 mol. 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 or LTQ) 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 Å˜150-220 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 &amp; Quantification. 
     Target protein (e.g. a phosphorylated proteins 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 or LTQ). 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 1×10 8 ; 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).