US 20100159477A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0159477 A1 Hornbeck et al. (43) Pub. Date: Jun. 24, 2010
(54) REAGENTS FOR THE DETECTION OF Publication Classification PROTEIN PHOSPHORYLATION IN (51) Int. Cl. SIGNALNG PATHWAYS GOIN 33/573 (2006.01) GOIN 33/53 (2006.01) (76) Inventors: Peter Hornbeck, Magnolia, MA C07K 6/00 (2006.01) C07K 7/06 (2006.01) (US); Valerie Goss, Seabrook, NH C07K 7/08 (2006.01) (US); Kimberly Lee, Seattle, WA C07K I4/00 (2006.01) (US); Ting-Lei Gu, Woburn, MA CI2N 5/071 (2010.01) (US); Albrecht Moritz, Salem, MA CI2N 5/16 (2006.01) (US) CI2N 5/18 (2006.01) (52) U.S. Cl...... 435/7.4; 435/7.1:530/387.1; 530/328; Correspondence Address: 530/327: 530/326; 530/325; 530/324; 435/326 Nancy Chiu Wilker, Ph.D. (57) ABSTRACT Chief Intellectual Property Counsel CELL SIGNALING TECHNOLOGY, INC., 3 The invention discloses novel phosphorylation sites identi Trask Lane fied in signal transduction proteins and pathways, and pro vides phosphorylation-site specific antibodies and heavy-iso Danvers, MA 01923 (US) tope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/ (21) Appl. No.: 12/309,311 proteins, as well as methods of using the reagents for Such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: adaptor/scaffold (22) PCT Filed: Jul. 13, 2007 proteins, adhesion/extracellular matrix protein, apoptosis proteins, calcium binding proteins, cell cycle regulation pro (86). PCT No.: PCT/US07f73537 teins, chaperone proteins, chromatin, DNA binding/repair/ replication proteins, cytoskeletal proteins, endoplasmic S371 (c)(1), reticulum or golgi proteins, enzyme proteins, G/regulator (2), (4) Date: Feb. 4, 2010 proteins, inhibitor proteins, motor/contractile proteins, phos phatase, protease, Ser/Thr protein kinases, Protein kinase (Tyr)s, receptor/channel/cell Suface proteins, RNA binding Related U.S. Application Data proteins, transcriptional regulators, tumor suppressor pro (60) Provisional application No. 60/830,724, filed on Jul. teins, ubiquitan conjugating system proteins and proteins of 13, 2006. unknown function.
crude protein (e.g., celi extractor cellular organelle) or in vitrokinase reaction or purified phosphoprotein
obtain protein fraction (e.g., lysis, organic extraction, detergent treatment, etc.) immobilize general protei modification-specific antibody to resin denature proteins (e.g., heat treatment)
digest cnide protein mixture to peptides N Y---- with immobilized trypsin of other protease contact digest with antibody-resi and incubate (e.g., 4, 1-6 hours)
remove unbound peptides by centrifugation, filtration, or colunt washing
wash antibody-resin extensively
eutebound peptides (e.g., 30% acetic acid, 0.1 M glycine, ph2.3, or 0.1%trifluoroacctic acid)
desalt and concetrate peptides with microcolumn (e.g., reversed-phase ZipTip) (optional)
analyze, e.g., MS for mass, MS after phosphatase treatenment to confinn phosphorylation, or tandem MS (MS/MS or MS) for partial sequence Patent Application Publication Jun. 24, 2010 Sheet 1 of 17 US 2010/01594.77 A1
FIGURE 1
crude protein (e.g., cell extract or cellular organelle) or in vitro kinase reaction or purified phosphoprotein
obtain protein fraction (e.g., lysis, organic extraction, detergent treatinent, etc.) immobilize general protein modification-specific antibody to resin denature proteins (e.g., heat treatment)
digest crude protein mixture to peptides with inmobilized trypsin or other protease w - contact digest with antibody-resin and incubate (e.g., 4-, 1-16 hours)
remove unbound peptides by cel ntrifugation, filtration, or column washing
Y wash antibody-resin extensively t elute bound peptides (e.g., 30% acetic acid, 0.1 M glycine, ph 2.3, or 0.1% trifluoroacetic acid) y desalt and concetrate peptides with microcolumn (e.g., reversed-phase ZipTip) (optional) w
analyze, e.g., MS for mass, MS after phosphatase treatenent to confirm phosphorylation, or tandem MS (MS/MS or MS) for partial sequence Patent Application Publication Jun. 24, 2010 Sheet 2 of 17 US 2010/01594.77 A1
Accession No. LSEEMADTLEEGSASPTSPDySLDSPGPEK
Adaptor scaffold Y942. KHEALMSDLSAySSSIQALR. QQVAPTDDEISKELVAyDYQEKSPR, Adaptor scaffold QTASDESyKDPTNIQSK Adaptor Scaffold 72 KMEEDyTNLSK
gz33 Adaptor? scaffold....Y56. YPELSQyMGLSLNEEEIR... normal human Adaptor Scaffold AEGPTIKNDASQQTNyGVAVLDKEQLSDYLK Adaptor scaffold LENOLyrcTOKYSPWGMK T Adaptor scaffold SLENOYTCTOKYSPWGMK II T cell leukemia ARyDFCAR it cell leukemia Adaptor scaffold WLECFEMALSNIRPDyADSNFHDFK daptor scaffold ARyDFCAR
NP-003248.3 Adaptor scaffold. Adhesion of extracellular . atric protein EKEPEEELyOLSKWLAGGVAPALFR Adhesion or extracellular matrix protein. PSPASVSVSAPAFyAPQKK. Cell cycle regulation. Y25 NLYPSSSyTR Cell cycle regulation. ANLQQAMTPLKFWDNTyYK.
lcycle regulation RDLIVRETVNVITLYK.
Cell cycle regulation YKYRDLTVRETVNVITLyK. Cell cycle regulation. GLlyGPPGTGK. . . .
QLlyiPPDEK NCWSIPTNSSK Patent Application Publication Jun. 24, 2010 Sheet 3 of 17 US 2010/01594.77 A1
Figure 2
Patient M4833
:ALCL 'AML B cell lymphoma: ECML :DLBCL terythroblasticacute leukemia breast cancer gastric cancer :lymphona 'melanoma s HLKEDOTEyLEER OState Cacer
aperone aperone Chaperone. SEQID NO. 34
Chaperone LDDAIEDCTNAWKLDDTyl K Chromatin, DNA-binding DNA repair or DNA replication NP.055925. protein Y282 romati?, DNA-binding, DNA repair or DNA replication protein Y1037 SMMSAyER NP-115571.1 Chromatin, DNA-binding, DNA repair or DNA replication P.11551.protein. SMMSSySAAOR NP 11557.1
repair of DNA replication Cotein . . . Chromatin, DNA-binding, DN repair or DNA replication 'protein Chromatin, DNA-binding, DN T cell ALL :repair of DNA replication
repair or DNA replication tei Chromatin, DNA-binding,BN celleukemi repair or DNA replication :NP 001062. 1: protein DMESDSGOGVDOLOR AML Patent Application Publication Jun. 24, 2010 Sheet 4 of 17 US 2010/01594.77 A1
Figure 2
Disease Telekefia colon cancer : isquamous cell Jurkat KFNHDGEEEEEDDDyGSR Carcinoma för?atiri, DNA-binding,D repair or DNA replication rotein
roteinhromatin, DNA-binding,- - - - DNA------DMESYYQEIGR. tein. Y289 RWSEDTSHyLIK. hromatin, DNA-binding, DNA : ------: :repair or DNA replication NPC64504.2 protein. Chromatin, DNA-binding. DNA repair or DNA replication
QVVKAELEKyOOYK......
DATNORKyLK KGWSLEDYTAFLDKESENK ytoskeletal protein
SISGPLSPAyIGQVPYNYNQLEGR, . SSISGPLSPAYTGQVPyNYNOLEGR
SEQD NO. 64
Patent Application Publication Jun. 24, 2010 Sheet 6 of 17 US 2010/01594.77 A1
Figure 2
icell Unel Tissue Patient SENO F302
001055. :NP 0010582
P-0041701:Enzyme, misc.
zyne, misc.Y.15. ... IcellALL. MOLT KG Zyne, misc. AWGEETDyTPWCMR LGNVIDPLDVyGISLQGLHNOLLNSNLDPSEVEKA zyme, misc.
LYDRAGEEHyNCSALHK ------m. m. SEQDNO.98
VFGHPFSLGWASPFATPDOGKADPyayW
GWFPANYWEEDySEYC.
Inhibitor protein WLECMLKKElyNK
:DQDELNPAAWR iT cell ALL MOLT15 Patent Application Publication Jun. 24, 2010 Sheet 7 of 17 US 2010/01594.77 A1
acute erythroblastic leukemia
Y1052 (DSSDIYNELKETYPNyLPLYWAR MOLT15 --- ALCL BaF34ZF MO-9 XP-371254.5 Y1597, GEVLEGSNAYCEK. MOLT15
NP-065116.2 ... Y312. CCMPIFICA. :AML s Icelleukemia
T celleukemia Jurkat SEQID NO.121
;HCC827 K562 MCF-10A(Y969F) : :MOL15 :Me-F2 K562 :Karpas-1105p Protein kinase, Seri Thr (non
NP061852.1 receptor) iy1215 Protein kinase, Seri Tir (non :NP_006385.2 receptor) Protein kinase, Tyr (non
celleukemia Jurka Patent Application Publication Jun. 24, 2010 Sheet 8 of 17 US 2010/0159477 A1
eceptor, channel, transporter surface protein. Y646. FVAWFVTVPWAGLFSAAVMALLMyGILPYV. cell surface protein. IAWFYIVPAGESAMALMYGLPY. Receptor, channel, transporter cell surface protein GSGSWGELuk ----- : eceptor, channel, transporter 2 or cell surface protein Y30 is spoEDssssssonseik :Receptor, channel, transporter iT celleukemia cell surface protein. AIGMFFSK. colon cance? ;Receptor, channel, transporter issy.goostock. VIVALARDSLALARPKSSDwyVSYDYGKSFK. MVALARDSLALARPKSSDVYvsyDYGKSFK. : ;Receptor, channel, transporter NP_001056.1 or cell surface protein Y318 REVAPPyaGADPLATALASDPIPNPLOK. :Receptor, channel, transporter DSAHKPQSLDFDDPATLyAWENVPPLR
ASLRDNLYLLQAFMylLGICL.
YPQPENPDSGGNYPRPKPR. RYDQDLCyTDILFTEOER T cell leukemia Jurkat snRNP 116 NP-004238.2 RNA binding protein BaF3
NAbinding protein NAbinding protein. AAFGISDSyWDGSSFDPQR QSDSSSyPTVDSNSLLGQSR. T cell leukemia squamous cell cinoma.
isPYSRSRyRESRYGGSHYSSSGYSNSR SEQID NO 161 Patent Application Publication Jun. 24, 2010 Sheet 9 of 17 US 2010/01594.77 A1
Figure 2
NAbinding protein Abinding protein. YGGDRGGGyGGDRGGYGGDRGGYGGDR.
NAbinding protein
RNA binding protein binding protein. NAbinding protein ENVEyEREESDGEYDEFGRK
ranscriptional regulator Scriptional regulator. fanscriptional regulator
ranscriptional regulator. scriptional regulator. ranscriptional regulator. scriptional regulator. ranscriptional regulator. PDGHGDYAyqQSSYTEQSYDRSF.
inscriptional regulator
LQDNFQEDPIQMSMySCLKEER. P909330.1 Transcriptional regulator CTV. KG-1 QFLAPWIESODWAyAASK MOLT15 DATNILVSPVYLyPDIPKEEAFGK Scriptional regulator PESQEHPEADPGSAAPYLK
LAVRQDVINTLLPK EALQSIATDPGLyoMLPR P055315.2 Transcriptional regulator. TQTDAGGEPDSLGQQPTDTPyEWDLDKK. NP 055315.2 Transcriptional requlator EFOEDSDEKEEEEDTyEK iT cell leukemia : Patent Application Publication Jun. 24, 2010 Sheet 10 of 17 US 2010/01594.77 A1
:NP003196.1 Transcriptional regulator. 2. SFIOSPHSCHNDSR. NP-003196.1 - QOLGLGSPAQLSSSGKPGTAyySFSATSSR P-0031961. Transcriptional regulator. GGSPAQLSSSGKPGTAYYSFSATSSR. NLTDyPIPR QWEKVETEyARYENGHYSYRIHR QWEKVETEYARyENGHYSYRIHR KVETEYARYENGHySYRIHR
CRPAASENyMR QLDKPASGVKEEWyAR. P859066.1 Y488. LPPYPySSPSLVLPIQPHTPK Scriptional regulator....Y228. SSKPWPDATyGTGSASR. ranscriptional regulator DGAySLFDNSK SSGFAFDPSVNySK. LNNEyEVIK.
HLGlySVLPNHK KHSADKykCK P_036614.1 Transcriptional regulator CDTCOOyFSR : Scriptional regulator ikNTDKNLNFVSPLPDVGok P.115765.2 anscriptional regulator P_0057172 Patent Application Publication Jun. 24, 2010 Sheet 11 of 17 US 2010/0159477 A1
cell lymphom ASNESIPGGyNALR. celleukemia CyCFNKPEDK. yTVQFTTMVQVNEETGNR PWSAGGEGETSPSLEASPLGOLMNMLSHPW
EMFNPMyALFR SVELPPYSGTVLCGTQAVDKLPDGQEyOR.
NP_0051421 Ubiquitin conjugating system. Y417. YEPFSFADDIGSNNCGyYDLQAVLTHQGR. iquitin conjugating system. Y239. NSNYCLPsyTAYK. Ubiquitin conjugating system. Y227. GyAQQDTQEFLR
in conjugating system. Y192YMSNTyEQLSK. 2 Ubiquitin conjugating syst SVTFDPFCyLILPLPLKK.
Ubiquitin conjugating system - - - - N EQWQSQR
:Unknown function... Y833. SPAySYSTEPR ... Unknown function Unknown function known function. TGQlyOPIQAY. Unknown function. 2. YSKEEEMDOMDRDLGDEyGWK
Unknown function - - - LKEAENKyDAIK known function KLALALENEGylK ZNF198FGFR. ------BaF34ZF :BaF3
Unknown function ------FLT3(D842Y). known function
Unknown function ------rain. SEQED NO 259
sunknown function.
Unknown function. ... Y180 ------ALL MOLT15. SEQIONO.261
Unknown function known function Unknown function known function
NP 0.0927.3:Unknown function Patent Application Publication Jun. 24, 2010 Sheet 12 of 17 US 2010/0159477 A1
Figure 2
IDyPCLLILDPQNEFEILR. FNVNTDyGNPVR TRPIQQFyOPPR HIGEKPyTCPDCGR. :Karpas 299 SR-786 nknown function NP-612472.1 Unknown function NF-6124721. Unknown function
Y1122 Sy(FGIGNDR celleukemia
known function. APQAIDGLAGALDAR Inknown function : WODWTATSAyk known function
QFLQyAYDITFDPDTAHKYRLQEENRK.
HNSDySYFK.
SPPPLPKDKNPPTPSIYLENK.
NAHSLAHSEESAMSNTMVNKDDLPVSKyYR P-060360.3 Unknown function. KNAHSLAHSEETSAMSNTMVNKDDLPWSKYyR :NP 0561932 Unknown function Patent Application Publication Jun. 24, 2010 Sheet 13 of 17 US 2010/0159477 A1
Figure 2
celleukemia C anaplastic
known function. Y456. TMTCEyALCSFFYFGDR
own function ------...... Sanlung unior, SEQID No. 310
AAEDSPyWSPAYSK EKIPDQMFFHTDYR
KETDPDyEDWSVALPNKR known function known function
MKNySQPMPLFDRSK. . . .
322 ZDHHC13 Unknown function ------cell leukemia ; AML known function AML B cell ALL :NSCLC T cell leukemia : colon cancer : imultiple NP_0562722 :Unknown function i GVGSPEPGPTAPyLGR myeloma :NPO37505.1. Unknown function. SQADSLFGDSGWDAPSSYSLOOASVLSEGPRGP
835461.1 Unknown function known function. 2 :RNEGWGGEDYEEVDRySR ":LKDEDFPSLSASTSSSCSTAATPGPWGLALPAPA P835461.1 :Unknown function Patent Application Publication Jun. 24, 2010 Sheet 14 of 17 US 2010/01594.77 A1
Figure 2
AML : iT cellALL Jurkat EMCF-10A(Y969F) : MOLT15 HRLoyKKKWNPDLOVEvK
NP008887.2 Unknown function PWGPEDMGATAWyELDTEKER T celleukemia Jurkat :NP_003909.1 unknown function. CM 293
DMTOCIGPNGyMEPYCSTACMNSHK PQy0ATMPDGKLYNFCNSSCVAK yeloproliferativ SLPOYQATMPDGKLyNFCNSSCVAK diseases DDyKKLHCVTYCEYCQEEK
myeloproliferativ:ZNF198FGFR 003441 iUnknown function CIWTYCEyCOEEK ediseases ZNF1987FGFR AML BaF3-10ZF CML BaF342F imyeloproliferativ :BaF3-APR :Unknown function. ediseases. known function nknown function. nknown function ATyCKPHMQTK . . . .
yTONTIQVAIOSLR T cell leukemia ; :293TTNT-FAf" ACL
NLSSDKyGDTSYHDEEEDEYEAEDDEEEEDEGRT cell leukemia Jurkat DTSyHDEEEDEYEAEDDEEEEDEGR. T celleukemia :
TYASGyAHYEECEN celleukemia :
NPOGOO65 1.1. Unknown function PFQCPECDKSyCIR 00107388 Unknown function NP00107388 ------0. Unknown function. Unknown function : SECONO. 360
Patent Application Publication Jun. 24, 2010 Sheet 16 of 17 US 2010/01594.77 A1
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REAGENTS FOR THE DETECTION OF to gain a finer appreciation of cellular regulation. In spite of PROTEIN PHOSPHORYLATION IN the importance of protein modification, phosphorylation is SIGNALNG PATHWAYS not yet well understood due to the extraordinary complexity of signaling pathways, and the slow development of the tech RELATED APPLICATIONS nology necessary to unravel it. 0008. In many instances, such knowledge is likely to pro 0001 Pursuant to 35 U.S.C. S119(e) this application vide valuable tools useful to evaluate, and possibly to claims the benefit of, and priority to, provisional application manipulate target pathways, ultimately altering the functional U.S. Ser. No. 60/830,724, filed Jul. 13, 2006, the disclosure of status of a given cell for a variety of purposes. which is incorporated herein, in its entirety, by reference. 0009. The importance of protein kinase-regulated signal TECHNICAL FIELD transduction pathways is underscored by a number of drugs designed to treat various cancer types by the inhibition of 0002 The invention relates generally to a variety of moi target protein kinases at the apex or intermediary levels of eties and tools for the detection of protein phosphorylation. pathways implicated in cancer development. See Sternet al., Moreover, the invention relates to the use of the same for Expert Opin. Ther. Targets 9(4):851-60 (2005). diagnostic and therapeutic purposes. 0010 Leukemia, a disease in which a number of underly ing signal transduction events have been elucidated, has BACKGROUND become a disease model for phosphoproteomic research and 0003. The activation of proteins by post-translational development efforts. As such, it represent a paradigm leading modification is an important cellular mechanism for regulat the way for many other programs seeking to address many ing most aspects of biological organization and control, classes of diseases (See, Harrison's Principles of Internal including growth, development, homeostasis, and cellular Medicine, McGraw-Hill, New York, N.Y.) communication. Cellular signal transduction pathways 0011 Depending on the cell type involved and the rate by involve protein kinases, protein phosphatases, and phosphop which the disease progresses leukemia can be defined as acute rotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3- or chronic myelogenous leukemia (AML or CML), or acute 3) containing cellular proteins to provide multidimensional, and chronic lymphocytic leukemia (ALL or CLL). dynamic and reversible regulation of many biological activi 0012 Most varieties of leukemia are generally character ties. See e.g., Sawyer et al., Med. Chem. 1(3): 293-319 ized by genetic alterations e.g., chromosomal translocations, (2005). deletions or point mutations resulting in the constitutive acti 0004 Protein phosphorylation on a proteome-wide scale Vation of protein kinase genes, and their products, particularly is extremely complex as a result of three factors: the large tyrosine kinases. The most well known alteration is the onco number of modifying proteins, e.g. kinases, encoded in the genic role of the chimeric BCR-Abl gene. See Nowell, Sci genome, the much larger number of sites on Substrate proteins ence 132: 1497 (1960)). The resulting BCR-Abl kinase pro that are modified by these enzymes, and the dynamic nature tein is constitutively active and elicits characteristic signaling of protein expression during growth, development, disease pathways that have been shown to drive the proliferation and states, and aging. The human genome, for example, encodes survival of CML cells (see Daley, Science 247: 824-830 over 520 different protein kinases, making them the most (1990); Raitano et al., Biochim. Biophys. Acta. December 9: abundant class of enzymes known. See Hunter, Nature 411: 1333(3): F201-16 (1997)). 355-65 (2001). Most kinases phosphorylate many different 0013 The recent success of Imanitib (also known as Substrate proteins, at distinct tyrosine, serine, and/or threo STI571 or GleevecR), the first molecularly targeted com nine residues. Indeed, it is estimated that one-third of all pound designed to specifically inhibit the tyrosine kinase proteins encoded by the human genome are phosphorylated, activity of BCR-Abl, provided critical confirmation of the and many are phosphorylated at multiple sites by different central role of BCR-Abl signaling in the progression of CML kinases. See Graves et al., Pharmacol. Ther: 82: 111-21 (see Schindleret al., Science 289; 1938-1942 (2000): Nardiet (1999). al., Curr. Opin. Hematol. 11:35-43 (2003)). 0005. Many of these phosphorylation sites regulate criti 0014. The success of GleevecR) now serves as a paradigm cal biological processes and may prove to be important for for the development of targeted drugs designed to block the diagnostic or therapeutic modalities useful in the treatment activity of other tyrosine kinases known to be involved in and management of many pathological conditions and dis many diseased including leukemias and other malignancies eases, including interalia cancer, developmental disorders, as (see, e.g., Sawyers, Curr. Opin. Genet. Dev. February; 12(1): as inflammatory, immune, metabolic and bone diseases. 111-5 (2002); Druker, Adv. Cancer Res. 91:1-30 (2004)). For 0006 For example, of the more than 100 dominant onco example, recent studies have demonstrated that mutations in genes identified to date, 46 are protein kinases. See Hunter, the FLT3 gene occur in one third of adult patients with AML. Supra. Understanding which proteins are modified by these FLT3 (Fms-like tyrosine kinase 3) is a member of the class III kinases will greatly expand our understanding of the molecu receptor tyrosine kinase (RTK) family including FMS, plate lar mechanisms underlying oncogenic transformation. There let-derived growth factor receptor (PDGFR) and c-KIT (see fore, the identification of, and ability to detect, phosphoryla Rosnet et al., Crit. Rev. Oncog 4:595-613 (1993). In 20-27% tion sites on a wide variety of cellular proteins is crucially of patients with AML, an internal tandem duplication in the important to understanding the key signaling proteins and juxta-membrane region of FLT3 can be detected (see Yokota pathways implicated in the progression of many disease et al., Leukemia 11: 1605-1609 (1997)). Another 7% of States. patients have mutations within the active loop of the second 0007 Understanding reversible protein phosphorylation kinase domain, predominantly Substitutions of aspartate resi and its role in the operation and interrelationship between due 835 (D835), while additional mutations have been cellular components and functions provides the opportunity described (see Yamamoto et al., Blood 97: 2434-2439 (2001): US 2010/0159477 A1 Jun. 24, 2010
Abu-Duhier et al., Br. J. Haematol. 113: 983-988 (2001)). at long last provides the elements necessary to attain those Expression of mutated FLT3 receptors results in constitutive much needed proteomics tools and modalities. tyrosine phosphorylation of FLT3, and Subsequent phospho 0020. The invention discloses novel phosphorylation sites rylation and activation of downstream molecules Such as identified in signal transduction proteins and pathways under STAT5, Akt and MAPK, resulting in factor-independent lying various disease states including for example human growth of hematopoietic cell lines. leukemias. The invention thus provides new reagents, includ 0015. Altogether, FLT3 is the single most common acti ing phosphorylation-site specific antibodies and AQUA pep vated gene in AML known to date. This evidence has trig tides, for the selective detection and quantification of these gered an intensive search for FLT3 inhibitors for clinical use phosphorylated sites/proteins. Also provided are methods of leading to at least four compounds in advanced stages of using the reagents of the invention for the detection and clinical development, including: PKC412 (by Novartis), quantification of the disclosed phosphorylation sites. CEP-701 (by Cephalon), MLN518 (by Millenium Pharma ceuticals), and SU5614 (by Sugen/Pfizer) (see Stone et al., BRIEF DESCRIPTION OF THE DRAWINGS Blood (in press)(2004); Smith et al., Blood 103: 3669-3676 0021 FIG. 1—Is a diagram broadly depicting the immu (2004); Clarket al., Blood 104:2867-2872 (2004); and Spiek noaffinity isolation and mass-spectrometric characterization erman et al., Blood 101: 1494-1504 (2003)). methodology (IAP) employed to identify the novel phospho 0016. There is also evidence indicating that kinases such rylation sites disclosed herein. as FLT3, c-KIT and Abl are implicated in some cases of ALL 0022 FIG. 2 Is a table (corresponding to Table 1) enu (see Cools et al., Cancer Res. 64: 6385-6389 (2004); Hu, Nat. merating the Leukemia signaling protein phosphorylation Genet. 36: 453-461 (2004); and Graux et al., Nat. Genet. 36: sites disclosed herein: Column A-the name of the parent 1084-1089 (2004)). In contrast, very little is know regarding protein; Column B-the SwissProt accession number for the any causative role of protein kinases in CLL, except for a high protein (human sequence); Column C the protein type/clas correlation between high expression of the tyrosine kinase sification; Column D-the tyrosine residue (in the parent pro ZAP70 and the more aggressive form of the disease (see tein amino acid sequence) at which phosphorylation occurs Rassenti et al., N. Eng. J. Med. 351: 893-901 (2004)). within the phosphorylation site; Column E=the phosphoryla 0017 Despite the identification of a few key molecules tion site sequence encompassing the phosphorylatable resi involved in progression of leukemia, the vast majority of due (residue at which phosphorylation occurs (and corre signaling protein changes underlying this disease remains sponding to the respective entry in Column D) appears in unknown. There is, therefore, relatively scarce information lowercase; Column F-the type of leukemia in which the about kinase-driven signaling pathways and phosphorylation phosphorylation site was discovered; and Column G=the cell sites relevant to the different types of leukemia. This has type(s), tissue(s) and/or patient(s) in which the phosphoryla hampered a complete and accurate understanding of how tion site was discovered. protein activation within signaling pathways is driving these 0023 FIG. 3 is an exemplary mass spectrograph depict complex cancers. Accordingly, there is a continuing and ing the detection of the tyrosine 786 phosphorylation site in pressing need to unravel the molecular mechanisms of TrkC (see Row 139 in FIG.2/Table 1), as further described in kinase-driven oncogenesis in leukemia by identifying the Example 1 (red and blue indicate ions detected in MS/MS downstream signaling proteins mediating cellular transfor spectrum);Y indicates the phosphorylated tyrosine (shown mation in this disease. Identifying particular phosphorylation as lowercase “y” in FIG. 2). sites on Such signaling proteins and providing new reagents, 0024 FIG. 4 is an exemplary mass spectrograph depict Such as phospho-specific antibodies and AQUA peptides, to ing the detection of the tyrosine 192 phosphorylation site in detect and quantify them remains particularly important to HSP90B (see Row 30 in FIG.2/Table 1), as further described advancing our understanding of the biology of this disease. in Example 1 (red and blue indicate ions detected in MS/MS 0018 Presently, diagnosis of leukemia is made by tissue spectrum);Y indicates the phosphorylated tyrosine (shown biopsy and detection of different cell surface markers. How as lowercase “y” in FIG. 2). ever, misdiagnosis can occur since some leukemia cases can 0025 FIG. 5 is an exemplary mass spectrograph depict be negative for certain markers, and because these markers ing the detection of the tyrosine 328 phosphorylation site in may not indicate which genes or protein kinases may be TOP2A (see Row 87 in FIG.2/Table 1), as further described deregulated. Although the genetic translocations and/or in Example 1 (red and blue indicate ions detected in MS/MS mutations characteristic of a particular form of leukemia can spectrum);Y indicates the phosphorylated serine (shown as be sometimes detected, it is clear that other downstream lowercase “y” in FIG. 2). effectors of constitutively active kinases having potential 0026 FIG. 6 is an exemplary mass spectrograph depict diagnostic, predictive, or therapeutic value, remain to be elu ing the detection of the tyrosine 15 phosphorylation site in cidated. Accordingly, identification of downstream signaling SNRPN (see Row 157 in FIG.2/Table 1), as further described molecules and phosphorylation sites involved in different in Example 1 (red and blue indicate ions detected in MS/MS types of leukemia and development of new reagents to detect spectrum);Y indicates the phosphorylated tyrosine (shown and quantify these sites and proteins may lead to improved as lowercase “y” in FIG. 2) diagnostic/prognostic markers, as well as novel drug targets, 0027 FIG. 7 is an exemplary mass spectrograph depict for the detection and treatment of this disease. ing the detection of the tyrosine 507 phosphorylation site in VPS35 (see Row 383 in FIG.2/Table 1), as further described SUMMARY OF THE INVENTION in Example 1 (red and blue indicate ions detected in MS/MS 0019. Several novel protein phosphorylation sites have spectrum);Y indicates the phosphorylated tyrosine (shown been identified in a variety of cell lines. Such novel phospho as lowercase “y” in FIG. 2). rylation sites (tyrosine), and their corresponding parent pro 0028 FIG. 8 is an exemplary mass spectrograph depict teins are reported (see Table 1). The elucidation of these sites ing the detection of the tyrosine 192 phosphorylation site in US 2010/0159477 A1 Jun. 24, 2010
TAGLN3 (see Row 66 in FIG.2/Table 1), as further described Column E. Row 2, of Table 1/FIG. 2 (which encompasses the in Example 1 (red and blue indicate ions detected in MS/MS phosphorylatable tyrosine at position 116). spectrum);Y indicates the phosphorylated tyrosine (shown 0033. In one embodiment, the invention provides an iso as lowercase “y” in FIG. 2). lated phosphorylation site-specific antibody that specifically binds a target signaling protein/polypeptide selected from DETAILED DESCRIPTION Column A of Table 1 (Rows 2-384) only when phosphory 0029 Several novel protein phosphorylation sites have lated at the tyrosine residue listed in corresponding ColumnD been identified in a variety of cell lines. Such novel phospho of Table 1, comprised within the phosphorylatable peptide rylation sites (tyrosine), and their corresponding parent pro sequence listed in corresponding Column E of Table 1 (SEQ teins are reported (see Table 1). The elucidation of these sites ID NOs: 1-383), wherein said antibody does not bind said at long last provides the elements necessary to attain those signaling protein when not phosphorylated at said tyrosine. In much needed proteomics tools and modalities. another embodiment, the invention provides an isolated phos 0030 The disclosure of the phosphorylation sites provides phorylation site-specific antibody that specifically binds a the key to the production of new moieties, compositions and target signaling protein/polypeptide selected from Column. A methods to specifically detect and/or to quantify these phos of Table 1 only when not phosphorylated at the tyrosine phorylated sites/proteins. Such moieties include for example residue listed in corresponding Column D of Table 1, com reagents. Such as phosphorylation site-specific antibodies and prised within the peptide sequence listed in corresponding AQUA peptides (heavy-isotope labeled peptides). Such Column E of Table 1 (SEQ ID NOs: 1-383), wherein said reagents are highly useful, interalia, for studying signal trans antibody does not bind said signaling protein when phospho duction events underlying the progression of many diseases rylated at said tyrosine. Such reagents enable the specific known or Suspected to involve protein phosphorylation e.g., detection of phosphorylation (or non-phosphorylation) of a leukemia in a mammal. Accordingly, the invention provides novel phosphorylatable site disclosed herein. The invention novel reagents phospho-specific antibodies and AQUA further provides immortalized cell lines producing such anti peptides—for the specific detection and/or quantification of a bodies. In one embodiment, the immortalized cell line is a target signaling protein/polypeptide (e.g., a signaling protein/ rabbit or mouse hybridoma. polypeptide implicated in leukemia) only when phosphory 0034. In another embodiment, the invention provides a lated (or only when not phosphorylated) at a particular phos heavy-isotope labeled peptide (AQUA peptide) for the quan phorylation site disclosed herein. The invention also provides tification of a target signaling protein/polypeptide selected methods of detecting and/or quantifying one or more phos from Column A of Table 1, said labeled peptide comprising phorylated target signaling protein/polypeptide using the the phosphorylatable peptide sequence listed in correspond phosphorylation-site specific antibodies and AQUA peptides ing Column E of Table 1 (SEQ ID NOs: 1-383), which of the invention. sequence comprises the phosphorylatable tyrosine listed in 0031. These phosphorylation sites correspond to numer corresponding Column D of Table 1. In certain embodiments, ous different parent proteins (the full sequences (human) of the phosphorylatable tyrosine within the labeled peptide is which are all publicly available in SwissProt database and phosphorylated, while in other embodiments, the phosphory their Accession numbers listed in Column B of Table 1/FIG. latable residue within the labeled peptide is not phosphory 2), each of which are have been linked to specific functions in lated. the literature and thus may be organized into discrete protein 0035 Reagents (antibodies and AQUA peptides) provided type groups, for example adaptor/scaffold proteins, cytoskel by the invention may conveniently be grouped by the type of etal proteins, protein kinases, and DNA binding proteins, etc. target signaling protein/polypeptide in which a given phos (see Column C of Table 1), the phosphorylation of which is phorylation site (for which reagents are provided) occurs. The relevant to signal transduction activity (e.g., underlying AML. protein types for each respective protein (in which a phos CML, CLL, and ALL), as disclosed herein. phorylation site has been discovered) are provided in Column 0032. In part, the invention provides an isolated phospho C of Table 1/FIG. 2, and include: adaptor/scaffold proteins, rylation site-specific antibody that specifically binds a given adhesion/extracellular matrix protein, apoptosis proteins, target signaling protein/polypeptide only when phosphory calcium binding proteins, cell cycle regulation proteins, lated (or not phosphorylated, respectively) at a particular chaperone proteins, chromatin, DNA binding/repair/replica tyrosine enumerated in Column D of Table 1/FIG. 2 com tion proteins, cytoskeletal proteins, endoplasmic reticulum or prised within the phosphorylatable peptide site sequence enu golgi proteins, enzyme proteins, G/regulator proteins, inhibi merated in corresponding Column E. In further part, the tor proteins, motor/contractile proteins, phosphatase, pro invention provides a heavy-isotope labeled peptide (AQUA tease, Ser/Thr protein kinases, protein kinase (Tyr)S, receptor/ peptide) for the detection and quantification of a given target channel/cell Suface proteins, RNA binding proteins, signaling protein/polypeptide, the labeled peptide compris transcriptional regulators, tumor Suppressor proteins, ubiq ing a particular phosphorylatable peptide site/sequence enu uitan conjugating system proteins and proteins of unknown merated in Column E of Table 1/FIG. 2 herein. For example, function. Each of these distinct protein groups is a Subset of among the reagents provided by the invention is an isolated target signaling protein/polypeptide phosphorylation sites phosphorylation site-specific antibody that specifically binds disclosed herein, and reagents for their detection/quantifica the VAV1 adaptor/scaffold protein only when phosphorylated tion may be considered a Subset of reagents provided by the (or only when not phosphorylated) at tyrosine 791 (see Row invention. 15 (and Columns D and E) of Table 1/FIG. 2). By way of 0036 Subsets of the phosphorylation sites (and their cor further example, among the group of reagents provided by the responding proteins) disclosed herein are those occurring on invention is an AQUA peptide for the quantification of phos the following protein types/groups listed in Column C of phorylated SLY adaptor/scaffold protein, the AQUA peptide Table 1/FIG. 2 adaptor/scaffold proteins, calcium binding comprising the phosphorylatable peptide sequence listed in proteins, chromatin or DNA binding/repair/replication pro US 2010/0159477 A1 Jun. 24, 2010 teins, cytoskeletal proteins, enzyme proteins, protein kinases Table 1 (SEQID NOS: 29-36), which sequence comprises the (Tyr), protein kinases (Ser/Thr), receptor/channel/trans phosphorylatable tyrosine listed in corresponding Column D. porter/cell Suface proteins, transcriptional regulators and Rows 30-37, of Table 1. translational regulators. Accordingly, among Subsets of 0041 Among this subset of reagents, antibodies and reagents provided by the invention are isolated antibodies and AQUA peptides useful for the detection and/or quantification AQUA peptides for the detection/quantification of the follow of the foregoing protein/phosphorylation site Subsets. ing chaperone protein phosphorylation sites are: HSP90B (Y 0037. The patents, published applications, and scientific 192), STI1 (Y269) and TPR2 (Y317) (see SEQID NOs: 29, literature referred to herein establish the knowledge of those 30 and 36). with skill in the art and are hereby incorporated by reference 0042 In another subset of embodiments there is provided: in their entirety to the same extent as if each was specifically (i) An isolated phosphorylation site-specific antibody that and individually indicated to be incorporated by reference. specifically binds a chromatin or DNA binding/repair/repli Any conflict between any reference cited herein and the spe cation protein selected from Column A. Rows 38-55, of Table cific teachings of this specification shall be resolved in favor 1 only when phosphorylated at the tyrosine listed in corre of the latter. Likewise, any conflict between an art-understood sponding Column D. Rows 38-55, of Table 1, comprised definition of a word or phrase and a definition of the word or within the phosphorylatable peptide sequence listed in corre phrase as specifically taught in this specification shall be sponding Column E. Rows 38-55, of Table 1 (SEQ ID NOs: resolved in favor of the latter. 37-54), wherein said antibody does not bind said protein 0038. In one subset of embodiments, there is provided: when not phosphorylated at said tyrosine. (i) An isolated phosphorylation site-specific antibody that (ii) An equivalent antibody to (i) above that only binds the specifically binds an cell cycle regulation protein selected chromatin or DNA binding/repair/replication protein when from Column A. Rows 23-29, of Table 1 only when phospho not phosphorylated at the disclosed site (and does not bind the rylated at the tyrosine listed in corresponding Column D. protein when it is phosphorylated at the site). Rows 23-29, of Table 1, comprised within the phosphorylat (iii) A heavy-isotope labeled peptide (AQUA peptide) for the able peptide sequence listed in corresponding Column E. quantification of a target signaling protein/polypeptide that is Rows 23-29, of Table 1 (SEQID NOs: 22-28), wherein said a chromatin or DNA binding/repair/replication protein antibody does not bind said protein when not phosphorylated selected from Column A. Rows 38-55, said labeled peptide at said tyrosine. comprising the phosphorylatable peptide sequence listed in (ii) An equivalent antibody to (i) above that only binds the cell corresponding Column E. Rows 38-55, of Table 1 (SEQID cycle regulation protein when not phosphorylated at the dis NOs: 37-54), which sequence comprises the phosphorylat closed site (and does not bind the protein when it is phospho able tyrosine listed in corresponding Column D. Rows 38-55, rylated at the site). of Table 1. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the 0043 Among this subset of reagents, antibodies and quantification of a cell cycle regulation protein selected from AQUA peptides for the detection/quantification of the follow Column A. Rows 23-29, said labeled peptide comprising the ing chromatin or DNA binding/repair/replication protein phosphorylatable peptide sequence listed in corresponding phosphorylation sites are: TOP2B (Y230), TSN (Y210), Column E. Rows 23-29, of Table 1 (SEQ ID NOS: 22-28), TYMS (Y153) and WRN (Y849) (see SEQID NO’s: 41,43, which sequence comprises the phosphorylatable tyrosine 46 and 50). listed in corresponding Column D. Rows 23-29, of Table 1. 0044 In still another subset of embodiments there is pro 0039. Among this subset of reagents, antibodies and vided: AQUA peptides for the detection/quantification of the follow (i) An isolated phosphorylation site-specific antibody that ing cell cycle regulation protein phosphorylation sites are: specifically binds a cytoskeletal protein selected from Col TSG 101 (Y32) and VCP (Y644) (see SEQ ID NOs: 25 and umn A. Rows 56-83, of Table 1 only when phosphorylated at 27). the tyrosine listed in corresponding Column D. Rows 56-83, 0040 In a second subset of embodiments there is pro of Table 1, comprised within the phosphorylatable peptide vided: sequence listed in corresponding Column E. Rows 56-83, of (i) An isolated phosphorylation site-specific antibody that Table 1 (SEQID NOs: 55-82), wherein said antibody does not specifically binds a chaperone protein selected from Column bind said protein when not phosphorylated at said tyrosine. A. Rows 30-37, of Table 1 only when phosphorylated at the (ii) An equivalent antibody to (i) above that only binds the tyrosine listed in corresponding Column D. Rows 30-37, of cytoskeletal protein when not phosphorylated at the disclosed Table 1, comprised within the phosphorylatable peptide site (and does not bind the protein when it is phosphorylated sequence listed in corresponding Column E. Rows 30-37, of at the site). Table 1 (SEQID NOS: 29-36), wherein said antibody does not (iii) A heavy-isotope labeled peptide (AQUA peptide) for the bind said protein when not phosphorylated at said tyrosine. quantification of a signaling protein that is a cytoskeletal (ii) An equivalent antibody to (i) above that only binds the protein selected from Column A. Rows 56-83, said labeled chaperone protein when not phosphorylated at the disclosed peptide comprising the phosphorylatable peptide sequence site (and does not bind the protein when it is phosphorylated listed in corresponding Column E. Rows 56-83, of Table 1 at the site). (SEQ ID NOs: 55-82), which sequence comprises the phos (iii) A heavy-isotope labeled peptide (AQUA peptide) for the phorylatable tyrosine listed in corresponding Column D. quantification of a target signaling protein/polypeptide that is Rows 56-83, of Table 1. a chaperone protein selected from Column A. Rows 30-37, 0045 Among this subset of reagents, antibodies and said labeled peptide comprising the phosphorylatable peptide AQUA peptides for the detection/quantification of the follow sequence listed in corresponding Column E. Rows 30-37, of ing cytoskeletal protein phosphorylation sites are: US 2010/0159477 A1 Jun. 24, 2010
SPTA1 (Y1538), SFTBN1 (Y1667), TAGLN3 (Y192), tubu 141, of Table 1, comprised within the phosphorylatable pep lin, beta-2 (Y51), VASP (Y16) and VIM (Y291) (see SEQID tide sequence listed in corresponding Column E. Rows 132 NOs: 56,60, 65, 74,78, and 80). 141, of Table 1 (SEQ ID NOs: 131-140), wherein said 0046. In still another subset of embodiments there is pro antibody does not bind said protein when not phosphorylated vided: at said tyrosine. (i) An isolated phosphorylation site-specific antibody that (ii) An equivalent antibody to (i) above that only binds the specifically binds an enzyme protein selected from Column protein kinase (Tyr) when not phosphorylated at the disclosed A. Rows 84-101, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D. Rows 84-101, of site (and does not bind the protein when it is phosphorylated Table 1, comprised within the phosphorylatable peptide at the site). sequence listed in corresponding Column E. Rows 84-101, of (iii) A heavy-isotope labeled peptide (AQUA peptide) for the Table 1 (SEQID NOs: 83-100), wherein said antibody does quantification of a signaling protein that is a protein kinase not bind said protein when not phosphorylated at said (Tyr) selected from Column A. Rows 132-141, said labeled tyrosine. peptide comprising the phosphorylatable peptide sequence (ii) An equivalent antibody to (i) above that only binds the listed in corresponding Column E. Rows 132-141, of Table 1 enzyme protein when not phosphorylated at the disclosed site (SEQ ID NOs: 131-140), which sequence comprises the (and does not bind the protein when it is phosphorylated at the phosphorylatable tyrosine listed in corresponding Column D. site). Rows 132-141, of Table 1. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the 0051. Among this subset of reagents, antibodies and quantification of a signaling protein that is a enzyme protein AQUA peptides for the detection/quantification of the follow selected from Column A. Rows 84-101, said labeled peptide comprising the phosphorylatable peptide sequence listed in ing protein kinase (Tyr) phosphorylation sites are: Yes corresponding Column E. Rows 84-101, of Table 1 (SEQID (Y146), TrkC (Y786) and Tyro3 (YG85) (see SEQID NOs: NOs: 83-100), which sequence comprises the phosphorylat 131, 138 and 139). able tyrosine listed in corresponding Column D. Rows 0.052 In yet another subset of embodiments, there is pro 84-101, of Table 1. vided: 0047 Among this subset of reagents, antibodies and (i) An isolated phosphorylation site-specific antibody that AQUA peptides for the detection/quantification of the follow specifically binds an RNA binding protein selected from Col ing enzyme protein phosphorylation sites are: TOP2A umn A. Rows 156-175, of Table 1 only when phosphorylated (Y328), TPH1 (Y401), TPI1 (Y48) and UAP1 (Y125) (see at the tyrosine listed in corresponding Column D. Rows 156 SEQID NOs: 86, 87, 89 and 91). 175, of Table 1, comprised within the phosphorylatable pep 0048. In still another subset of embodiments there is pro tide sequence listed in corresponding Column E. Rows 156 vided: 175, of Table 1 (SEQ ID NOs: 155-174), wherein said (i) An isolated phosphorylation site-specific antibody that antibody does not bind said protein when not phosphorylated specifically binds a protein kinase (Ser/Thr) selected from at said tyrosine. Column A. Rows 123-131, of Table 1 only when phosphory (ii) An equivalent antibody to (i) above that only binds the lated at the tyrosine listed in corresponding Column D. Rows RNA binding protein when not phosphorylated at the dis 123-131, of Table 1, comprised within the phosphorylatable closed site (and does not bind the protein when it is phospho peptide sequence listed in corresponding Column E. Rows rylated at the site). 123-131 of Table 1 (SEQ ID NOs: 122-130), wherein said antibody does not bind said protein when not phosphorylated (iii) A heavy-isotope labeled peptide (AQUA peptide) for the at said tyrosine. quantification of a signaling protein that is a RNA protein (ii) An equivalent antibody to (i) above that only binds protein selected from Column A. Rows 156-175, said labeled peptide kinase (Ser/Thr) when not phosphorylated at the disclosed comprising the phosphorylatable peptide sequence listed in site (and does not bind the protein when it is phosphorylated corresponding Column E. Rows 156-175, of Table 1 (SEQID at the site). NOs: 155-174), which sequence comprises the phosphorylat (iii) A heavy-isotope labeled peptide (AQUA peptide) for the able tyrosine listed in corresponding Column D. Rows 156 quantification of a signaling protein that is a protein kinase 175, of Table 1. (Ser/Thr) selected from Column A. Rows 123-131, said 0053 Among this subset of reagents, antibodies and labeled peptide comprising the phosphorylatable peptide AQUA peptides for the detection/quantification of the follow sequence listed in corresponding Column E. Rows 123-131, ing RNA protein phosphorylation sites are: SNRPN (Y15), of Table 1 (SEQ ID NOs: 122-130), which sequence com UPF2(Y974) and UPF3B (Y160) (see SEQID NOs: 156, 169 prises the phosphorylatable tyrosine listed in corresponding and 170). Column D, Rows 123-131, of Table 1. 0054 In yet another subset of embodiments, there is pro 0049 Among this subset of reagents, antibodies and vided: AQUA peptides for the detection/quantification of the follow (i) An isolated phosphorylation site-specific antibody that ing protein kinase (Ser/Thr) phosphorylation sites are: PKCD specifically binds a transcriptional regulator selected from (Y374) and TRRAP (Y3497) (see SEQID NO: 122 and 128). Column A. Rows 176-231, of Table 1 only when phosphory 0050. In yet another subset of embodiments, there is pro lated at the tyrosine listed in corresponding Column D. Rows vided: 176-231, of Table 1, comprised within the phosphorylatable (i) An isolated phosphorylation site-specific antibody that peptide sequence listed in corresponding Column E. Rows specifically binds a protein kinase (Tyr) selected from Col 176-231, of Table 1 (SEQ ID NOs: 175-230), wherein said umn A. Rows 132-141, of Table 1 only when phosphorylated antibody does not bind said protein when not phosphorylated at the tyrosine listed in corresponding Column D. Rows 132 at said tyrosine. US 2010/0159477 A1 Jun. 24, 2010
(ii) An equivalent antibody to (i) above that only binds the tide sequence listed in corresponding Column E of Table 1 transcriptional regulator when not phosphorylated at the dis (SEQID NO: 4), which sequence comprises the phosphory closed site (and does not bind the protein when it is phospho latable tyrosine listed in corresponding Column D. Row 5 of rylated at the site). Table 1. (iii) A heavy-isotope labeled peptide (AQUA peptide) for the 0059. The invention also provides an immortalized cell quantification of a target signaling protein/polypeptide that is line producing an antibody of the invention, for example, a a transcriptional regulator selected from Column A. Rows cell line producing an antibody within any of the foregoing 176-231, said labeled peptide comprising the phosphorylat subsets of antibodies. In an embodiment, the immortalized able peptide sequence listed in corresponding Column E. cell line is a rabbit hybridoma or a mouse hybridoma. Rows 176-231, of Table 1 (SEQ ID NOs: 175-230), which 0060. In other embodiments, a heavy-isotope labeled pep sequence comprises the phosphorylatable tyrosine listed in tide (AQUA peptide) of the invention (for example, an AQUA corresponding Column D. Rows 176-231, of Table 1. peptide within any of the foregoing Subsets of AQUA pep 0055 Among this subset of reagents, antibodies and tides) comprises a disclosed site sequence wherein the phos AQUA peptides for the detection/quantification of the follow phorylatable tyrosine is phosphorylated. In yet other embodi ing transcriptional regulator phosphorylation sites are: ments, a heavy-isotope labeled peptide of the invention comprises a disclosed site sequence wherein the phosphory SPT5 (Y140), SSB (Y23), SSRP1 (Y452), STAT3 (YG74), latable tyrosine is not phosphorylated. STAT5B (Y171), TAF172 (Y415), TCF12 (Y82), TEL 0061 The foregoing subsets of reagents of the invention (Y401) and TFIIF (Y124) (see SEQ ID NO: 178, 185, 186, should not be construed as limiting the scope of the invention, 190, 192, 194, 201, 211 and 213). which, as noted above, includes reagents for the detection 0056 In still another subset of embodiments, there is pro and/or quantification of disclosed phosphorylation sites on vided: any of the other protein type/group Subsets (each a Subset) (i) An isolated phosphorylation site-specific antibody that listed in Column C of Table 1/FIG. 2. specifically binds a translational regulator selected from Col 0062 Also provided by the invention are methods for umn A. Rows 234-249, of Table 1 only when phosphorylated detecting or quantifying a target signaling protein/polypep at the tyrosine listed in corresponding Column D. Rows 234 tide that is tyrosine phosphorylated, said method comprising 249, of Table 1, comprised within the phosphorylatable pep the step of utilizing one or more of the above-described tide sequence listed in corresponding Column E. Rows 234 reagents of the invention to detect or quantify one or more 249, of Table 1 (SEQ ID NOs: 233-248), wherein said target Signaling Protein(s)/Polypeptide(s) selected from Col antibody does not bind said protein when not phosphorylated umn A of Table 1 only when phosphorylated at the tyrosine at said tyrosine. listed in corresponding Column D of Table 1. In certain (ii) An equivalent antibody to (i) above that only binds the embodiments of the methods of the invention, the reagents translational regulator when not phosphorylated at the dis comprise a Subset of reagents as described above. The anti closed site (and does not bind the protein when it is phospho bodies according to the invention maybe used in standard rylated at the site). (e.g., ELISA or conventional cytometric assays). The inven (iii) A heavy-isotope labeled peptide (AQUA peptide) for the tion thus, provides compositions and methods for the detec quantification of a signaling protein that translational regula tion and/or quantitation of a given target signaling protein or tor selected from Column A, Rows 234-249, said labeled polypeptide in a sample, by contacting the sample and a peptide comprising the phosphorylatable peptide sequence control sample with one or more antibody of the invention listed in corresponding Column E. Rows 234-249, of Table 1 under conditions favoring the binding and thus formation of (SEQ ID NOS: 233-248), which sequence comprises the the complex of the antibody with the protein or peptide. The phosphorylatable tyrosine listed in corresponding Column D. formation of the complex is then detected according to meth Rows 234-249, of Table 1. ods well established and known in the art. 0057. Among this subset of reagents, antibodies and 0063 Also provided by the invention is a method for AQUA peptides for the detection/quantification of the follow obtaining a phosphorylation profile of a certain protein type ing a translational regulator phosphorylation sites are: USP14 or group, for example adaptor/scaffold proteins or cell cycle (Y417) and USP20 (Y227) (see SEQID NO: 243 and 245). regulation proteins (Rows 2-20 and Rows 23-29, respectively, 0058. In yet a further subset of embodiments, there is of Table 1), that is phosphorylated in a disease signaling provided: pathway, said method comprising the step of utilizing one or (i) An isolated phosphorylation site-specific antibody that more isolated antibody that specifically binds the protein specifically binds SPTAN1 (Y976), (Column A. Row 5, of group selected from Column A of Table 1 only when phos Table 1) only when phosphorylated at the tyrosine listed in phorylated at the tyrosine listed in corresponding Column D. corresponding ColumnD of Table 1), said tyrosine comprised of Table 1, comprised within the phosphorylation site within the phosphorylatable peptide sequence listed in corre sequence listed in corresponding Column E, to detect the sponding Column E of Table 1 (SEQID NO: 4), wherein said phosphorylation of one or more of said protein group, thereby antibody does not bind said protein when not phosphorylated obtaining a phosphorylation profile for said protein group. at said tyrosine. 0064. The invention further contemplates compositions, (ii) An equivalent antibody to (i) above that only binds foremost pharmaceutical compositions, containing onr or a SPTAN1 (Y976) (Column A. Row 5 of Table 1) when not more antibody according to the invention formulated together phosphorylated at the disclosed site (and does not bind the with a pharmaceutically acceptable carrier. One of skill will protein when it is phosphorylated at the site). appreciate that in certain instances the composition of the (iii) A heavy-isotope labeled peptide (AQUA peptide) for the invention may further comprise other pharmaceutically quantification of SPTAN1 (Y976) (Column A. Row 5 of Table active moieties. The compounds according to the invention 1), said labeled peptide comprising the phosphorylatable pep are optionally formulated in a pharmaceutically acceptable US 2010/0159477 A1 Jun. 24, 2010
vehicle with any of the well-known pharmaceutically accept continue a particular treatment dose, mode of administration, able carriers, including diluents and excipients (see Reming etc. The term “therapeutic composition” refers to any com ton's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack pounds administered to treat or prevent a disease. It will be Publishing Co., Easton, Pa. 1990 and Remington: The Sci understood that the Subject to which a compound (e.g., an ence and Practice of Pharmacy, Lippincott, Williams & antibody) of the invention is administered need not suffer Wilkins, 1995). While the type of pharmaceutically accept from a specific traumatic state. Indeed, the compounds (e.g., antibodies) of the invention may be administered prophylac able carrier/vehicle employed in generating the compositions tically, prior to any development of symptoms. The term of the invention will vary depending upon the mode of admin “therapeutic.” “therapeutically, and permutations of these istration of the composition to a mammal, generally pharma terms are used to encompass therapeutic, palliative as well as ceutically acceptable carriers are physiologically inert and prophylactic uses. Hence, as used herein, by “treating or non-toxic. Formulations of compositions according to the alleviating the symptoms” is meant reducing, preventing, invention may contain more than one type of compound of the and/or reversing the symptoms of the individual to which a invention), as well any other pharmacologically active ingre compound of the invention has been administered, as com dient useful for the treatment of the symptom/condition being pared to the symptoms of an individual receiving no Such treated. administration. 0065. The invention also provides methods of treating a 0067. The term “therapeutically effective amount” is used mammal comprising the step of administering Such a mam to denote treatments at dosages effective to achieve the thera mal a therapeutically effective amount of a composition peutic result sought. Furthermore, one of skill will appreciate according to the invention. that the therapeutically effective amount of the compound of 0066. As used herein, by “treating is meant reducing, the invention may be lowered or increased by fine tuning preventing, and/or reversing the symptoms in the individual and/or by administering more than one compound of the to which a compound of the invention has been administered, invention, or by administering a compound of the invention as compared to the symptoms of an individual not being with another compound. See, for example, Meiner, C. L., treated according to the invention. A practitioner will appre “Clinical Trials. Design, Conduct, and Analysis,' Mono ciate that the compounds, compositions, and methods graphs in Epidemiology and Biostatistics, Vol.8 Oxford Uni described herein are to be used in concomitance with con versity Press, USA (1986). The invention therefore provides tinuous clinical evaluations by a skilled practitioner (physi a method to tailor the administration/treatment to the particu cian or veterinarian) to determine Subsequent therapy. Hence, lar exigencies specific to a given mammal. As illustrated in following treatment the practitioners will evaluate any the following examples, therapeutically effective amounts improvement in the treatment of the pulmonary inflammation may be easily determined for example empirically by starting according to standard methodologies. Such evaluation will at relatively low amounts and by step-wise increments with aid and inform in evaluating whether to increase, reduce or concurrent evaluation of beneficial effect.
TABL E 1 Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEO ID NO 2 SLY NPO 61863. 1 Adaptor/scaffold Y116 ALSEEMADTLEEGSASPTSPDySLDSPGPEK SEO ID NO. 1
3 SPTAN1 NPOO311.8.1 Adaptor/scaffold Y6O1 TATDEAyKDPSNLQGK SEO ID NO. 2
4 SPTAN1 NPOO311.8.1 Adaptor/scaffold Y942 KHEALMSDLSAyOSSIQALR SEO ID NO. 3
5 SPTAN1 NPOO3118.1 Adaptor/scaffold Y976 QQVAPTDDETGKELVLALyDYQEKSPR SEO ID NO. 4
6 SPTAN1 NPOO311.8.1 Adaptor/scaffold Y1O2O QGFVPAAyVK SEQ NO. 5
7 SPTAN1 NPOO311.8.1 Adaptor/scaffold Y1579 LOTASDESyKDPTNIQSK SEQ NO 6
8 SYNE2 NP O55995.4 Adaptor/scaffold Y3372 KMEEDIyTNLSK SEQ NO f
9 synergin, NPOO91783 Adaptor/scaffold Y514 ALPSMDKyAVFK SEQ NO 8 gamma
10 syntenin NPOO5616.2 Adaptor/scaffold Y56 LYPELSQyMGLSLNEEEIR SEQ NO. 9
11 TRAF3IP3 NPO795O4.1 Adaptor/scaffold Y165 AEGPTIKNDASQQTNyGVAVLDKEIIQLSDYLK SEQ NO. 10
12 TRAF3IP3 NPO795O4.1 Adaptor/scaffold Y234 SLENOLyTCTOKYSPWGMK SEQ NO. 11
13 TRAF3IP3 NPO795O4.1 Adaptor/scaffold Y24 O SLENOLYTCTOKySPWGMK SEQ NO. 12
14 TRAF3IP3 NPO795O4.1 Adaptor/scaffold Y302 SQyEALKEDWR SEQ NO. 13 US 2010/0159477 A1 Jun. 24, 2010
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 15. WAW1 NPOO5419.2 Adaptor/scaffold Y791 ARyDFCAR SEQ ID NO. 14
16. WAW3 NPOO 6104.4 Adaptor/scaffold Y508 WLEQFEMALSNIRPDyADSNFHDFK SEQ ID NO. 15 17 WAV3 NPOO 6104.4 Adaptor/scaffold Y797 ARyDFCAR SEQ ID NO. 16
18 WAC NPO5771.2.2 Adaptor/scaffold Y25 RGDSQPyOALK SEO ID NO. 17 19 ZO1 NPOO3248.3 Adaptor/scaffold Y830 ADGATSDDLDLHDDRLSYLSAPGSEySMYSTDSR SEQ ID NO. 18 2O ZO1 NPOO3248.3 Adaptor/scaffold Y1324 TLyRIPEPOKPOLKPPEDIVR SEQ ID NO. 19
21URP2 NP 113659.3 Adhesion or Y162 EKEPEEELyDLSKVVLAGGVAPALFR SEQ ID NO. 20 extracellular matrix protein 22 ZyXin NPOO3452.1 Adhesion or Y2O PSPAISVSVSAPAFyAPQKK SEQ ID NO. 21 extracellular matrix protein
23 SUGT1 NP OO6695.1 Ce cycle Y251 NLYPSSSPyTR SEQ ID NO. 22 regulation
24 TPX2 NPO3 6.24 4.2 Ce cycle Y81 ANLQQAIVTPLKPVDNTyYK SEQ ID NO. 23 regulation
25 TSG101 NPOO6283. Ce cycle Y15. yKYRDLTVRETVNVITLYK SEQ ID NO. 24 regulation
26 TSG101 NPOO 62.83. Ce cycle Y32 YKYRDLTVRETVNVITLyK SEQ ID NO. 25 regulation
27WCP NPOO9057. Ce cycle Y244 GILLyGPPGTGK SEQ ID NO. 26 regulation
28 WCP NPOO9057. Ce cycle Y644 LDQLIyIPLPDEK SEO ID NO. 27 regulation
29 ZW10 NPOO4715. Ce cycle Y349 NCLVySIPTNSSK SEQ ID NO. 28 regulation
3OHSP9 OB NPO31381.2 Chaperone Y192 VILHLKEDOTEyLEER SEQ ID NO. 29
31STI1 NPOO6810. Chaperone Y269 ELDPTNMTYITNOAAVyFEKGDYNK SEQ ID NO. 30
32 STI1 NPOO 681O. Chaperone Y27s ELDPTNMTYITNOAAVYFEKGDyNK SEQ ID NO. 31
33 STI1 NPOO6810. Chaperone Y444 AAALEAMKDyTK SEQ ID NO. 32
34 TBCA NPOO4598. Chaperone Y48 MRAEDGENyDIKK SEQ ID NO. 33
35 TBCA NPOO4598. Chaperone Y94 ILENEKDLEEAEEyKEAR SEQ ID NO. 34
36 TPR2 NPOO33 O6. Chaperone Y41 KDYNEAYNYyTK SEO ID NO. 35
37TPR2 NPOO33 O6. Chaperone Y317 KLDDAIEDCTNAVKLDDTyIK SEQ ID NO. 36
38 Smcs NP O55925. Chromatin, DNA- Y282 RPWVEYENVRQEyEEVK SEO ID NO. 37 binding, DNA repair or DNA replication protein
39 SON NP 115571.1 Chromatin, DNA- Y1037 SMMSAyER SEQ ID NO. 38 binding, DNA repair or DNA replication protein US 2010/0159477 A1 Jun. 24, 2010
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 4 OSON NP 115571.1 Chromatin, DNA- Y1096 SMMSSySAADR SEQ ID NO. 39 binding, DNA repair or DNA replication protein
41. SON NP 115571.1 Chromatin, DNA- Y2.192 EADSVyGEWVPVEK SEQ ID NO. 40 binding, DNA repair or DNA replication protein
42 TOP2B NPOO1059.2 Chromatin, DNA- Y23 O IKHFDGEDy TCITFQPDLSK SEQ ID NO. 41 binding, DNA repair or DNA replication protein
43 TOP2B NPOO1059.2 Chromatin, DNA- Y344 HVDyVVDQVVGK SEQ ID NO. 42 binding, DNA repair or DNA replication protein
44 TSN NPOO 46.13.1 Chromatin, DNA- Y210 KVEEVVyDLSIR SEQ ID NO. 43 binding, DNA repair or DNA replication protein
45 TSNAX NPOO5990.1 Chromatin, DNA- Y237 QVYDGFSFIGNTGPyEVSKK SEQ ID NO. 44 binding, DNA repair or DNA replication protein
46 TTF2 NPOO3585.3 Chromatin, DNA- Y285 GGPLNKEyTNWEAK SEQ ID NO. 45 binding, DNA repair or DNA replication protein
47 TYMS NPOO1062. 1 Chromatin, DNA- Y153 DMESDySGQGWDQLQR SEQ ID NO. 46 binding, DNA repair or DNA replication protein
48 UKp68 NPO791OO .. 2 Chromatin, DNA- Y321 KFNHDGEEEEEDDDyGSR SEO ID NO. 47 binding, DNA repair or DNA replication protein
49 WBP11 NP O57396.1 Chromatin, DNA- Y236 RRDEDMLySPELAQR SEQ ID NO. 48 binding, DNA repair or DNA replication protein
SOWBP11 NP O57396.1 Chromatin, DNA- Y630 DDVyEAFMK SEQ ID NO. 49 binding, DNA repair or DNA replication protein US 2010/0159477 A1 Jun. 24, 2010 10
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 5.1 WRN NPOOO544.2 Chromatin, DNA- Y849 DMESYyQEIGR SEO ID NO. 50 binding, DNA repair or DNA replication protein
52 XPG NPOOO114.2 Chromatin, DNA- Y289 RVVSEDTSHyILIK SEQ ID NO. 51 binding, DNA repair or DNA replication protein
53 ZAP NP 0645 04. 2 Chromatin, DNA- Y299 KFTyLGSQDR SEQ ID NO. 52 binding, DNA repair or DNA replication protein
54 ZAP NPO 645 O4.2 Chromatin, DNA- Y348 FLENGSQEDLLHGNPGSTyLASNSTSAPNWK SEO ID NO. 53 binding, DNA repair or DNA replication protein
55 ZAP NP 0645 04. 2 Chromatin, DNA- Y826 DAIySHKNCPYDAK SEQ ID NO. 54 binding, DNA repair or DNA replication protein
56 SPIRE1 NPO 64.533.2 Cytoskeleta Y4 OS QVLVKAELEKyOQYK SEO ID NO. 55 protein
57 SPTA1 NPOO3117.2 Cytoskeleta Y1.538 DATNIQRKyLK SEO ID NO. 56 protein
58 SPTA1 NPOO3117.2 Cytoskeleta Y2333 KGyVSLEDYTAFLIDKESENIK SEO ID NO. 57 protein
59 SPTBN1 NP 842565. 2 Cytoskeleta Y66 ITDLyTDLR SEO ID NO. 58 protein
6OSPTBN1 NP 8425 65.2 Cytoskeleta Y129 O MLTAQDMSyDEAR SEO ID NO. 59 protein
61 SPTBN1 NP 8425 65.2 Cytoskeleta Y1667 VDKLyAGLKDLAEER SEQ ID NO. 60 protein
62 SPTBN1 NP 8425 65.2 Cytoskeleta Y17 TSSISGPLSPAyTGQVPYNYNQLEGR SEQ ID NO. 61 iso 2 protein
63 SPTBN1 NP 8425652 Cytoskeleta Y23 TSSISGPLSPAYTGQVPyNYNQLEGR SEQ ID NO. 62 iso 2 protein
64 SPTBN2 NPOO 8877.1 Cytoskeleta Y1726 EVVAASHELGODyEHVTMLR SEQ ID NO. 63 protein
65 SSFA2 NP OO6742.2 Cytoskeleta Y930 NSLQNLSQyPMMR SEQ ID NO. 64 protein
66 TAGLN3 NPO37391.2 Cytoskeleta Y192 GASQAGMTGyGMPR SEO ID NO. 65 protein
67talin 1 NPOO628O. 2 Cytoskeleta Y71 KGIWLEAGKALDYyMLR SEQ ID NO. 66 protein
68 tau. NPOO59 O1.2 Cytoskeleta Y197 SGySSPGSPGTPGSR SEO ID NO. 67 protein
US 2010/0159477 A1 Jun. 24, 2010 13
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
124 TAO1 NP O65842.1 Protein kinase, Y43 EIGHGSFGAVyFAR SEQ ID NO. 123 Ser/Thr (non receptor)
125TAO2 NPOO4774.1 Protein kinase, Y61 KMSysGKQSNEK SEQ ID NO. 124 Ser/Thr (non receptor)
126 Titin NPOO331 O.3 Protein kinase, Y4912 PIEDVTIyEK SEQ ID NO. 125 Ser/Thr (non receptor)
127 Tilt in NPOO331 O.3 Protein kinase, Y15976 ITGyIVEK SEQ ID NO. 126 Ser/Thr (non receptor)
128 TRRAP NPOO3487.1 Protein kinase, Y862 TLELCVDNLOPDFLyDHIQPVR SEQ ID NO. 127 Ser/Thr (non receptor)
129 TRRAP NPOO3487.1 Protein kinase, Y3497 GHNGKIyPYLVMNDACLTESR SEQ ID NO. 128 Ser/Thr (non receptor)
13 OWNK1 NPO 61852.1 Protein kinase, Y1215 DDyGFSGSQK SEQ ID NO. 129 Ser/Thr (non receptor)
131 YSK1 NPOO 63 65.2 Protein kinase, Y35 IGKGSFGEVyK SEQ ID NO. 13 O Ser/Thr (non receptor)
132 Yes NPOO5424.1 Protein kinase, Y146 NGYIPSNyVAPADSIQAEEWYFGK SEQ ID NO. 131 Tyr (non receptor)
133 ZAP70 NPOO1070.2 Protein kinase, Y178 KLySGAQTDGK SEQ ID NO. 132 Tyr (non receptor)
134 ZAP70 NPOO1070.2 Protein kinase, Y2O4 KEQGTYALSLIyGK SEQ ID NO. 133 Tyr (non receptor)
135 ZAP70 NPOO1070.2 Protein kinase, Y597 MRACyYSLASK SEQ ID NO. 134 Tyr (non receptor)
136 ZAP70 NPOO1070.2 Protein kinase, Y598 MRACYySLASK SEQ ID NO. 135 Tyr (non receptor)
37 TIE1 NPOO5415.1 Protein kinase, Y1O83 NCDDEVyELMR SEQ ID NO. 136 Tyr (receptor)
38 TrkC NPOO2521.2 Protein kinase, Y558 VFLAECyNLSPTKDK SEO ID NO. 137 Tyr (receptor)
39 TrkC NPOO2521.2 Protein kinase, Y786 EVyDVMLGCWOR SEQ ID NO. 138 Tyr (receptor) 40 Tyro3 NPOO6284. 2 Protein kinase, Y685 KIYSGDyYROGCASKLPVK SEQ ID NO. 139 Tyr (receptor)
41 Tyro3 NPOO 6284.2 Protein kinase, Y686 KIYSGDYyROGCASKLPVK SEQ ID NO. 14 O Tyr (receptor)
42 SLC2OA2 NP OO674O1 Receptor, Y423 LVGDTVSySK SEQ ID NO. 141 channel, US 2010/0159477 A1 Jun. 24, 2010 14
TABLE 1 - continued Phosphorylation Sites
D Protein B C Phospho- E 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO transporter or cell surface protein
14.3 SLC2OA2 NP OO6740. Receptor, Y646 NIFVAWFVTVPVAGLFSAAVMALLMyGILPYV SEQ ID NO. 142 channel, transporter or cell surface protein
144 SLC2OA2 NP OO6740. Receptor, Y651 NIFVAWFVTVPVAGLFSAAVMALLMYGILPyv SEQ ID NO. 143 channel, transporter or cell surface protein
145 SLC25A12 NPOO3696. Receptor, GSGSVVGELMyK SEQ ID NO. 144 channel, transporter or cell surface protein
146 SLC38A2 NPO 61849. Receptor, FSISPDEDSSSYSSNSDFNYSyPTK SEQ ID NO. 145 channel, transporter or cell surface protein
147 SLC4Af NPOO3606. Receptor, SEQ ID NO. 146 channel, transporter or cell surface protein
148 SNX6 Receptor, Y1.37 IGSSLyALGTQDSTDICK SEQ ID NO. 147 channel, transporter or cell surface protein
149 SORL1 Receptor, Y136 SNVIVALARDSLALARPKSSDVyVSYDYGKSFK SEQ ID NO. 148 channel, transporter or cell surface protein
15OSORL1 Receptor, Y139 SNVIVALARDSLALARPKSSDVYVSyDYGKSFK SEQ ID NO. 149 channel, transporter or cell surface protein
151 TNF-R1 NPOO 1056. Receptor, Y318 REVAPPyOGADPILATALASDPIPNPLOK SEQ ID NO. 15 O channel, transporter or cell surface protein
152 TNF-R1 NP 001056. Receptor, Y360 WEDSAHKPQSLDTDDPATLyAVVENVPPLR SEQ ID NO. 151 channel, transporter or cell surface protein
15.3 TRPM3 NPO 66 OO3. Receptor, Y250 INESLRDQLLVTIQKTFTy SEQ ID NO. 152 channel, transporter or cell surface protein US 2010/0159477 A1 Jun. 24, 2010 15
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 15.4 TSPAN15 NPO3 6471.1 Receptor, Y97 ASLRDNLYLLQAFMyILGICL SEQ ID NO. 153 channel, transporter or cell surface protein
155 XG NP 78O778.1 Receptor, Y52 PPyYPOPENPDSGGNIYPRPKPR SEQ ID NO. 154 channel, transporter or cell surface protein
56 snRNP1.16 NPOO4238.2 RNA binding Y167 KRYDODLCy TDILFTEQER SEO ID NO. 155 protein
57 SNRPN NPOO3O88.1 RNA binding Y15. MLQHIDyR SEQ ID NO. 156 protein
58 SRim3 OO NPO57417.3 RNA binding Y14.5 AAFGISDSyVDGSSFDPQR SEO ID NO. 157 protein
59 SRim3 OO NPO57417.3 RNA binding Y1145 FQSDSSSyPTVDSNSLLGQSR SEQ ID NO. 158 protein
6OSRim3 OO NP O57417.3 RNA binding Y239 O. VPLSAyER SEO ID NO. 159 protein
61 SRp46 NP 115285. RNA binding Y17s SPySRSRYRESRYGGSHYSSSGYSNSR SEQ ID NO. 16 O protein
62 SRp46 NP 115285. RNA binding Y18O SPYSRSRyRESRYGGSHYSSSGYSNSR SEQ ID NO. 161 protein
63 SRp46 NP 115285. RNA binding Y190 SPYSRSRYRESRYGGSHySSSGYSNSR SEQ ID NO. 162 protein
64 STAU2 NPO552O8. RNA binding Y388 GILHLSPDVyQEMEASR SEQ ID NO. 163 protein
65 TAF15 NPOO3478. RNA binding Y67 SQSYGGyENQKQSSY SEQ ID NO. 164 protein
66 TAF15 NPOO3478. RNA binding Y491 GGYGGDRGGGyGGDRGGYGGDRGGYGGDR SEQ ID NO. 165 protein
67 TARDBP NPO314 O1. RNA binding Y155 FTEyETOVK SEQ ID NO. 166 protein
68 US-2OOkD NPO54733.2 RNA binding Y1682 IHAYVDYPIyDVLOMVGHANRPLODDEGR SEO ID NO. 167 protein
69 US-2OOkD NP O54733.2 RNA binding Y1770 RMTONPNyYNLQGISHR SEQ ID NO. 168 protein
7 OUPF2 NP O56357.1 RNA binding Y974 KSLEVWTKDHPFPIDIDyMISDTLELLR SEQ ID NO. 169 protein
71UPF3B NP O75386.1 RNA binding Y160 VGTIDDDPEyR SEO ID NO. 17 O protein
72 UPF3B NP O75386.1 RNA binding Y429 NKDRPAMQLyOPGAR SEQ ID NO. 171 protein
73 ZNF265 NPOO5446.2 RNA binding Y1O2 TGyGGGFNER SEQ ID NO. 172 protein
74 ZNF265 NPOO5446.2 RNA binding Y114 ENVEyIEREESDGEYDEFGRK SEO ID NO. 173 protein US 2010/0159477 A1 Jun. 24, 2010 16
TABL E 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ NO 75 ZNF265 NPOO54 46.2 RNA binding Y124 ENVEYIEREESDGEyDEFGRK SEQ NO. 74 protein
76 SMARCE1 Transcriptiona Y142 AYHNSPAYLAyINAK SEQ NO. regulator
77 SMRT NPOO 63 O3.3 Transcriptiona Y2249 SAVyPLLYR SEQ NO. 76 regulator
78 SND1 NPO552O5.2 Transcriptiona Y113 TPQGREYGMIyLGKDTNGENIAESLVAEGLATR SEQ NO. 77 regulator
79 SPTs NPOO3160.2 Transcriptiona Y14 O DOREEELGEyYMK SEQ NO. 78 regulator
8OSPTs NPOO 316O2 Transcriptiona Y295 GIYKDDIAQVDyVEPSONTISLK SEQ NO. 79 regulator
81 SPTs NPOO 316O2 Transcriptiona Y778 TPMYGSQTPMyGSGSR SEQ NO. regulator
82 SPTs NPOO 316O2 Transcriptiona TPMyGSQTPLODGSR SEQ NO. 81 regulator
83 SS NP 945173. Transcriptiona Y272 yPDGHGDYAYQQSSYTEQSYDRSF SEQ NO. 82 regulator
84 SS18L1 NP 9451.73. Transcriptiona Y281 YPDGHGDYAyQQSSYTEQSYDRSF SEQ NO. 83 regulator
85 SS NP 9451.73. Transcriptiona Y286 YPDGHGDYAYQQSSyTEQSYDRSF SEQ NO. 84 regulator
86 SSB NPOO3133. Transcriptiona Y23 ICHQIEyYFGDFNLPR SEQ NO. 85 regulator
87 SSRP1 NPOO3137. Transcriptiona EGMNPSYDEYADSDEDOHDAyLER SEQ NO. 86 regulator
88 STAT1 NPOO933O. Transcriptiona RNLQDNFQEDPIOMSMIIySCLKEER SEQ NO. 87 regulator
89 STAT1 NPOO933O. Transcriptiona Y668 YLyPNIDKDHAFGK SEQ NO. 88 regulator
9 OSTAT3 NP 64 48 Os. Transcriptiona QFLAPWIESQDWAyAASK SEQ NO. 89 regulator
91 STAT3 NP 64 48 Os. Transcriptiona Y674 IMDATNILVSPLVYLyPDIPKEEAFGK SEQ NO. 9 O regulator
92 STAT3 NP 644805. Transcriptiona ycRPESQEHPEADPGSMPYLK SEQ NO. 91 regulator
93 STATSB Transcriptiona Y171 SEQ NO. 92 regulator
94 STATSB Transcriptiona YyTPVPCESATAK SEQ NO. 93 regulator
95 TAF172 NPOO3963. 1 Transcriptiona Y415 yALAVRQDVINTLLPK SEQ NO. 94 regulator
96 TAF6 NPOO5632.1 Transcriptiona Y253 RAEALQSIATDPGLyOMLPR SEQ NO. 95 regulator
97 TAT-SE1 NP O55315.2 ranscriptiona YO DGDTOTDAGGEPDSLGOOPTDTPyEWDLDKK SEQ NO. 96 regulator US 2010/0159477 A1 Jun. 24, 2010 17
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 198TAT-SF1 NPO55315.2 Transcriptiona Y634 EFDEDSDEKEEEEDTyEK SEO ID NO. 197 regulator
199 TAT-SF1 NPO55315.2 Transcriptiona Y650 VFDDESDEKEDEEyADEK SEQ ID NO. 198 regulator
2 OOTCERG1 NPOO6697.2 Transcriptiona Y149 TPDGKVYyYNARTR SEQ ID NO. 199 regulator
2O1 TCERG1 NPOO6697.2 Transcriptiona Y150 TPDGKVYYyNARTR SEQ ID NO. 200 regulator
2O2 TCF12 NPOO3196.1 Transcriptiona Y82 GFTDSPHySDHLNDSR SEQ ID NO. 201 regulator
2O3 TCF12 NPOO31961 Transcriptiona Y156 QDLGLGSPAQLSSSGKPGTAyYSFSATSSR SEQ ID NO. 202 regulator
2O4 TCF12 NPOO31961 Transcriptiona Y157 QDLGLGSPAQLSSSGKPGTAYySFSATSSR SEQ ID NO. 203 regulator
2O5 TCF2O NPOO5641.1 Transcriptiona Y839 QINLTDyPIPR SEQ ID NO. 204 regulator
2O6 TEF-3 NPOO32O4. 2 Transcriptiona Y349 QVVEKVETEyARYENGHYSYRIHR SEQ ID NO. 205 regulator
2. Of TEF-3 NPOO32O4.2 Transcriptiona Y352 QVVEKVETEYARyENGHYSYRIHR SEQ ID NO. 206 regulator
2O8 TEF-3 NPOO32O4.2 Transcriptiona Y357 QVVEKVETEYARYENGHySYRIHR SEO ID NO. 207 regulator
209 Te NPOO1978. Transcriptiona Y6 O LQPIyWSRDDVAQWLK SEQ ID NO. 208 regulator
210 Te NPOO1978. Transcriptiona Y114 YRSPHSGDVLyELLOHILK SEQ ID NO. 209 regulator
211 Te NP OO1978. Transcriptiona Y346 LLWDYVyOLLSDSR SEQ ID NO. 210 regulator
212 Te NPOO1978. Transcriptiona Y4 O1 HyYKLNIIR SEQ ID NO. 211 regulator
213 Te NPOO1978. Transcriptiona Y4 O2 HYyKLNIIR SEQ ID NO. 212 regulator
214 TFIIF NPOO 4119. Transcriptiona Y124 AECRPAASENyMR SEQ ID NO. 213 regulator
21.5 TFII-I NP OO15 O9.2 Transcriptiona Y419 EDLQLDKPASGVKEEWyAR SEQ ID NO. 214 regulator
216 TORC2 NP 859066.1 Transcriptiona Y488 LPPYPySSPSLVLPTOPHTPK SEQ ID NO. 215 regulator
217 TRAP150 NPOO5110.1 Transcriptiona Y228 ASESSKPWPDATyGTGSASR SEQ ID NO. 216 regulator
218 Trap170 NPOO4220.2 Transcriptiona Y941 DGAySLFDNSK SEQ ID NO. 217 regulator
219 TRIM22 NPOO6065. 2 Transcriptiona Y394 SSGFAFDPSVNySK SEQ ID NO. 218 regulator
22 OTRIP11 NPOO4230.1 Transcriptiona Y452 LNNEyEVIK SEQ ID NO. 219 regulator US 2010/0159477 A1 Jun. 24, 2010 18
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 221 TRIP11 NPOO 423 O. 1 Transcriptiona Y1267 LQVDyTGLIQSYEQNETK SEQ ID NO. 22 O regulator
222 TSC22D2 NPO55594.1 Transcriptiona Y7 O3 SHLMyAVR SEQ ID NO. 221 regulator
223 UXT NPOO 4173.1 Transcriptiona Y43 DKVyEQLAK SEQ ID NO. 222 regulator
224 YY1 NPOO3394.1 Transcriptiona Y251 DIDHETVVEEQIIGENSPPDySEYMTGK SEQ ID NO. 223 regulator
225 YY1 NPOO3394.1 Transcriptiona Y254 DIDHETVVEEQIIGENSPPDYSEyMTGK SEQ ID NO. 224 regulator
226 ZBTB16 NPOO5997.2 Transcriptiona Y334 HLGIySVLPNHK SEQ ID NO. 225 regulator
227 ZNF141 NPOO3432.1 Transcriptiona Y423 KIHSADKPyKCK SEQ ID NO. 226 regulator
228 ZNF281 NPO3 6614.1 Transcriptiona Y353 CDTCQQyFSR SEO ID NO. 227 regulator
229 ZNF281 NPO3 6614.1 Transcriptiona Y479 KNTDKNyLNFVSPLPDIVGOK SEQ ID NO. 228 regulator
23 OZNF289 NP 115765.2 Transcriptiona Y278 LAyOELQIDRK SEQ ID NO. 229 regulator
231 ZNF33A NPOO8885.1 Transcriptiona Y45 DVMLENySNLVSVGYCVHK SEQ ID NO. 23 O regulator
232 TSFM NPOO57172 Translational Y59 TGySFVNCK SEQ ID NO. 231
233 TUFFM N POO3312.3 Translational Y269 DLEKPFLLPVEAVySVPGR SEQ ID NO. 232
234 UBC3B NPO60281.2 Ubiquitin Y190 VPTTLAEyCIK SEQ ID NO. 233 conjugating system
235 UBCEFIP3 NPOO6453.1 Ubiquitin Y220 QQQQEGNyLOHVOLDQR SEQ ID NO. 234 conjugating system
236 UBE1L, NPOO3326.2 Ubiquitin Y15. LLDEELySR SEQ ID NO. 235 conjugating system 237 ubiquilin2 NP O38472.2 Ubiquitin Y265 ALSNLESIPGGyNALR SEQ ID NO. 236 conjugating system 238 ubiquit in NPOO2945.1 Ubiquitin Y148 CCLTyCFNKPEDK SEO ID NO. 237 conjugating system
239 UREB1 NP 113584.3 Ubiquitin Y1658 YTVQFTTMVOVNEETGNR SEQ ID NO. 238 conjugating system
24 OUREB1 NP 113584.3 Ubiquitin Y3424 SVPVSAGGEGETSPySLEASPLGQLMNMLSHPVI SEQ ID NO. 239 conjugating RR system
241 UREB1 NP 113584.3 Ubiquitin Y4 O78 EMFNPMyALFR SEQ ID NO. 24 O conjugating system US 2010/0159477 A1 Jun. 24, 2010 19
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
242 USP10 NPOO5144.2 Ubiquitin Y54 SSVELPPYSGTVLCGTQAVDKLPDGQEyQR SEQ ID NO. 241 conjugating system
243 USP11 NPOO 4642.2 Ubiquitin Y396 SQFLGyQQHDSQE SEQ ID NO. 242 conjugating system
244 USP14 NPOO514.2.1 Ubiquitin Y417 YEPFSFADDIGSNNCGyYDLQAVLTHOGR SEQ ID NO. 243 conjugating system
245 USP15 NPOO 63 O4.1 Ubiquitin Y239 NSNYCLPsyTAYK SEQ ID NO. 244 conjugating system
246 USP2O NPOO6667.2 Ubiquitin Y227 GyAQODTOEFLR SEQ ID NO. 245 conjugating system
247 USP4 NPOO3354.2 Ubiquitin Y192 YMSNTyEOLSK SEQ ID NO. 246 conjugating system
248 USP4 NPOO3354.2 Ubiquitin Y476 VSVTFDPFCyLTLPLPLKK SEQ ID NO. 247 conjugating system
249 WWP2 NPOO8945.2 Ubiquitin Y369 NyEQWQSQR SEQ ID NO. 248 conjugating system
250SLFNS XP 4962O6.1 Unknown function Y115 AEVENKGySYK SEQ ID NO. 249
251 SLITRKS NPO 563 82.1 Unknown function Y833 SPAySVSTIEPR SEQ ID NO. 250
252 SMAP-5 NP 11 O426.4 Unknown function Y26 SIDDQSQQSyDYGGSGGPY SEQ ID NO. 251
253 SMAP-5 NP 110426. 4 Unknown function Y28 SIDDQSQQSYDyGGSGGPY SEQ ID NO. 252
254 SMAP-5 NP 11 O426.4 Unknown function Y67 TGQIyOPTQAY SEQ ID NO. 253
255 SMBP NP 0645 O8.2 Unknown function Y272 YSKEEEMDDMDRDLGDEyGWK SEQ ID NO. 254
256 SMC6L1 NPO789 OO. Unknown function Y780 SLKIEAENKyDAIK SEO ID NO. 255
257 SMEK2 NP O65196. Unknown function Y171 EKLALALENEGylK SEQ ID NO. 256
258 SNF8 NPOO9172.2 Unknown function Y192 NGyVTVSEIK SEO ID NO. 257
259 SNX14 NP O652O1. Unknown function Y353 SLHEELQKIyKTY SEQ ID NO. 258
26 OSPATA2 NPOO 6O29. Unknown function Y257 SVDAyDSYWESR SEO ID NO 259
261 SPATA2 NPOO6O29. Unknown function Y260 SVDAYDSyWESR SEQ ID NO. 26 O
262 SPATA5 NP. 66 O2 O8. Unknown function Y18O IVLPGNFLycTFYGRPYK SEQ ID NO. 261
263 SPATA5 NP. 66 O2 O8. Unknown function Y793 IIyVPLPDAATRR SEQ ID NO. 262
254 SSX9 NP 777622. Unknown function Y48 SSEKIIyVYMKR SEQ ID NO. 263
265 SSX9 NP 777622. Unknown function Y50 SSEKIIYVyMKR SEQ ID NO. 264
266 STS-1 NP 1162 62.2 Unknown function Y402 MDVVFGKyWLSQCFDAK SEQ ID NO. 265
267 SYAP1 NP 116.185.2 Unknown function Y60 DFGNyLFNFASAATK SEQ ID NO. 266
268 SYAP1 NP 116.185.2 Unknown function Y327 ELQQELQEyEVVTESEKR SEO ID NO. 267 US 2010/0159477 A1 Jun. 24, 2010 20
TABLE 1 - continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO
269 SYNPO NPOO9217.3 Unknown function Y222 MSGRAAATTPTKVySE SEQ ID NO. 268
27 OTBC1D12 XPO51O 81. 4 Unknown function Y800 LyEDILLQMDFIHIAQFLTK SEQ ID NO. 269
271 TBC1D13 NPO6 O671.2 Unknown function Y143 ATDyPCLLILDPONEFETLR SEO ID NO. 27 O
272 TDRD3 NP 11 O421.1 Unknown function Y481 HFNVNTDyONPVR SEQ ID NO. 271
273 TDRD3 NP 11 O421.1 Unknown function Y644 STRPTOQFyoPPR SEO ID NO. 272
274 TIP2O NPOO10O2836.1. Unknown function Y150 IHTGEKPyTCPDCGR SEO ID NO. 273
275 TIPRL NP 69 O866.1 Unknown function Y209 LYHEADKTyMLR SEQ ID NO. 274
276 TLCD1 NP 612472.1 Unknown function Y173 VNKyVNLVMYFLFR SEO ID NO. 275
277 TLCD1 NP 612472.1 Unknown function Y179 VNKYVNLVMyFLFR SEO ID NO. 276
278 TNKS1BP1 NP 203754.2 Unknown function Y855 DSQGTySSRDAELQDQEFGKR SEO ID NO. 277
279 TNKS1BP1 NP 203754.2 Unknown function Y940 DVSLGTyGSR SEO ID NO. 278
28O TNKS1BP1 NP 203754.2 Unknown function Y1122 SyOFGIIGNDR SEO ID NO. 279
281TNRC6B NPO559 O3.1 Unknown function Y1081 DMGTTDSGPyFEKGGSHGLFGNSTAQSR SEQ ID NO. 28O
282 TNRC6B NPO559 O3.1 Unknown function Y1388 GIONIDPESDPyVTPGSVLGGTATSPIVDTDHQL SEQ ID NO. 281 LR
283 TOM1 NPOO5479.1 Unknown function Y386 yEAPQATDGLAGALDAR SEQ ID NO. 282
284 TPD52 NPOO5070.1 Unknown function Y96 GWODVTATSAyK SEQ ID NO. 283
285 TPD52L2 NPOO3279.2 Unknown function Y1O6 SWHDVQVSSAyVK SEQ ID NO. 284
286 TPR18 NP. 66 O153.2 Unknown function Y1O23 EYEKAKKTyMOACK SEQ ID NO. 285
287 TRIM16 NPOO 6461.3 Unknown function Y371 EQFLOyAYDITFDPDTAHKYLRLQEENRK SEQ ID NO. 286
288 TRIM34 NPO67629.2 Unknown function Y391 yRPLFGYWVIGLONKCK SEO ID NO. 287
289 TRIM34 NPO67629.2 Unknown function Y397 YRPLFGyWVIGLONKCK SEQ ID NO. 288
29 OTRIMS NP 44321.O. 1 Unknown function Y524 TGVSPySKTLVLQTSEGK SEQ ID NO. 289 isos
291. TRS85 NP O55754.2 Unknown function Y179 IOHNSDySYPK SEQ ID NO. 290
292 TRS85 NPO55754.2 Unknown function Y181 IOHNSDYSyPK SEQ ID NO. 291
293 TSC22D2 NP O55594.1 Unknown function Y57 ATDyGPEEVCER SEQ ID NO. 292
294 TTC21B NPO79029.3 Unknown function Y160 GKEPyTKKALK SEQ ID NO. 293
295 UBAP2 NP 060919.2 Unknown function Y199 FSTOGMGTFNPADySDSTSTDVCGTK SEQ ID NO. 294
296 UBAP2 NPO6 O919.2 Unknown function Y1111 SQASKPAyGNSPYWTN SEO ID NO. 295
297 UBAP2 NPO6 O919.2 Unknown function Y1116 SQASKPAYGNSPyWTN SEQ ID NO. 296
298 USH3A NP 443721.1 Unknown function Y95 HLSEKIANy KEGTY SEO ID NO. 297
2.99 USP 47 BAB55O 631 Unknown function Y81 EGSVGSTSDyVSQSYSYSSILNK SEQ ID NO. 298
3 OO USP 47 BAB55O 631 Unknown function Y86 EGSVGSTSDYVSQSySYSSILNK SEQ ID NO. 299
3 O1 UTX NPO 66963. 1 Unknown function Y928 PPSSPyPPLPKDKLNPPTPSIYLENK SEQ ID NO. 3OO
3 O2 WPS13B NPO6 O3 6O.3 Unknown function Y242 LHFTyENLNSK SEQ ID NO. 301
US 2010/0159477 A1 Jun. 24, 2010 23
TABLE 1- Continued Phosphorylation Sites
A. D Protein B C Phospho- E H 1. Name Accession No. Protein Type Residue Phosphorylation Site Sequence SEQ ID NO 371 ZNF609 NPO55857.1 Unknown function Y796 AEADKIySFTDNAPSPSIGGSSR SEO ID NO. 37 O
372 ZNF622 NP 219482.1 Unknown function Y284 DHSFFIPDIEyLSDIK SEO ID NO. 371
373 ZSWIM4 NPO75560.1 Unknown function Y548 yLFTALLPHDPDLAYRLALR SEO ID NO. 372
374 SNAP29 NPOO4773.1 Vesicle protein Y160 yOASHPNLR SEO ID NO. 373 375 SNX1 NPOO3O90.2 Vesicle protein Y162 IGDGMNAyVAYK SEO ID NO. 374
376 SNX2 NPOO3 O91.2 Vesicle protein Y2O3 YLHVGyIVPPAPEK SEO ID NO. 375 377 SNX27 NP 11218O. 4 Vesicle protein Y186 FVVYNVyMAGR SEO ID NO. 376 378 STX16 NPOO3754.2 Vesicle protein Y66 WVDGVDEIOyDVGR SEO ID NO. 377 379 STXBPS NP 64 O337.2 Vesicle protein Y502 NKDDRPNTDIVDEDPyAIQIISWCPESR SEO ID NO. 378 380SW2A NPO55664. 2 Vesicle protein Y65 FEEEDDDDDFPAPSDGyYR SEO ID NO. 379 381 SW2A NP O55664. 2 Vesicle protein Y66 FEEEDDDDDFPAPSDGYyR SEQ ID NO. 38O
382 WPS29 NPO57310.1 Vesicle protein Y69 GDFDENLNyPEOK SEQ ID NO. 381 383 WPS35 NP O6 O676.2 Vesicle protein Y507 SEDPDQQyLILNTAR SEQ ID NO. 382 384 WPS4B NPOO4860. 2 Vesicle protein Y40 AGNYEEALQLYQHAVQyFLHVVK SEQ ID NO. 383
0068. The short name for each protein in which a phos- two light (L) chains. Antibodies possess two distinct and phorylation site has presently been identified is provided in spatially separate functional features. The ends of each of the Column A, and its SwissProt accession number (human) is two arms of the “Y” contain the variable regions (variable provided Column B. The protein type/group into which each heavy (V(H)) and variable light (V(L)) regions), which form protein falls is provided in Column C. The identified tyrosine two identical antigen-binding sites. The variable regions residue at which phosphorylation occurs in a given protein is undergo a process of “affinity maturation” during the immune identified in Column D, and the amino acid sequence of the response, leading to a rapid divergence of amino acids within phosphorylation site encompassing the tyrosine residue is these variable regions. The other end of the antibody mol provided in Column E (lower case y=the tyrosine (identified ecule, the stem of the “Y”, contains only the two heavy in Column D)) at which phosphorylation occurs. Table 1 constant (CH) regions, interacts with effector cells to deter above is identical to FIG. 2, except that the latter includes the mine the effector functions of the antibody. There are five disease and cell type(s) in which the particular phosphoryla different CH region genes that encode the five different tion site was identified (Columns F and G). classes of immunoglobulins: IgM, Ig), IgG, IgA and IgE. 0069) “Antibody” or “antibodies” refers to all classes of These constant regions, by interacting with different effector immunoglobulins, including IgG, IgM, IgA, Ig|D, and IgE, cells and molecules, determine the immunoglobulin mol including whole antibodies and any antigen biding fragment ecule's biological function and biological response. thereof (e.g., F.) or single chains thereof, including chi 0071. Each V(H) and V(L) region contains three subre meric, polyclonal, and monoclonal antibodies. Antibodies are gions called complementarity determining regions. These antigen-specific protein molecules produced by lymphocytes include CDR1-3 of the V(H) domain and CDR1-3 of the V(L) of the B cell lineage. Following antigenic stimulation, B cells domain. These six CDRS generally form the antigen binding that have surface immunoglobulin receptors that bind the Surface, and include those residues that hypermutate during antigen clonally expand, and the binding affinity for the anti the affinity maturation phase of the immune response. The gen increases through a process called affinity maturation. CDR3 of the V(H) domain seems to play a dominant role in The B cells further differentiate into plasma cells, which generating diversity oof both the B cell antigen receptor secrete large quantities of antibodies in to the serum. While (BCR) and the T cell antigen receptor systems (Xu et al., the physiological role of antibodies is to protect the host Immunity 13:37-45 (2000)). animal by specifically binding and eliminating microbes and (0072. The term “antibody” or “antibodies” refers to all microbial pathogens from the body, large amounts of anti classes of polyclonal or monoclonal immunoglobulins, bodies are also induced by intentional immunization to pro including IgG, IgM, IgA, Ig), and IgE, including whole duce specific antibodies that are used extensively in many antibodies and any antigen binding fragment thereof. This biomedical and therapeutic applications. includes any combination of immunoglobulin domains or 0070 Antibody molecules are shaped somewhat like the chains that contains a variable region (V(H) or V(L)) that letter “Y”, and consist of 4 protein chains, two heavy (H) and retains the ability to bind the immunogen. Such fragments US 2010/0159477 A1 Jun. 24, 2010 24 include F(ab) fragments (V(H)-C(H1), V(L)-C(L)); described as having values between 0 and 2, can be 0, 1 or 2 monovalent Fab fragments (V(H)-C(H1), V(L)-C(L)). Fv for variables which are inherently discrete, and can be 0.0, fragment (V(H)-V(L); single-chain Fv fragments (Kobayashi 0.1, 0.01, 0.001, or any other real value for variables which et al., Steroids July; 67(8):733-42 (2002). are inherently continuous. 0.073 Monoclonal antibodies refer to clonal antibodies 0078. As used in this specification, whether in a transi produced from fusions between immunized murine, rabbit, tional phrase or in the body of the claim, the terms “comprise human, or other vertebrate species, and produced by classical (s)' and "comprising are to be interpreted as having an fusion technology Kohler G. Milstein C. Continuous cultures open-ended meaning. That is, the terms are to be interpreted of fused cells secreting antibody of predefined specificity. synonymously with the phrases “having at least” or “includ Nature 1975 Aug. 7:256(55.17):495-7 or by alternative meth ing at least'. When used in the context of a process, the term ods which may isolate clones of immunoglobulin secreting “comprising means that the process includes at least the cells from transformed plasma cells. recited steps, but may include additional steps. When used in 0074. When used with respect to an antibody's binding to the context of a compound or composition, the term "com one phospho-form of a sequence, the expression “does not prising means that the compound or composition includes at bind’ means that a phospho-specific antibody either does not least the recited features or components, but may also include apparently bind to the non-phospho form of the antigen as additional features or components. ascertained in commonly used experimental detection sys (0079) “Antibody” or “antibodies' refers to all classes of tems (Western blotting, IHC. Immunofluorescence, etc.). immunoglobulins, including IgG, IgM, IgA, Ig), and IgE, One of skill in the art will appreciate that the expression may including whole antibodies and any antigen biding fragment be applicable in those instances when (1) a phospho-specific thereof (e.g., F.) or single chains thereof, including chi antibody either does not apparently bind to the non-phospho meric, polyclonal, and monoclonal antibodies. Antibodies are form of the antigen as ascertained in commonly used experi antigen-specific protein molecules produced by lymphocytes mental detection systems (Western blotting, IHC. Immunof of the B cell lineage. Following antigenic stimulation, B cells luorescence, etc.); (2) where there is some reactivity with the that have surface immunoglobulin receptors that bind the Surrounding amino acid sequence, but that the phosphory antigen clonally expand, and the binding affinity for the anti lated residue is an immunodominant feature of the reaction. gen increases through a process called affinity maturation. In cases Such as these, there is an apparent difference in The B cells further differentiate into plasma cells, which affinities for the two sequences. Dilutional analyses of Such secrete large quantities of antibodies in to the serum. While antibodies indicates that the antibodies apparent affinity for the physiological role of antibodies is to protect the host the phosphorylated form is at least 10-100 fold higher than for animal by specifically binding and eliminating microbes and the non-phosphorylated form; or where (3) the phospho-spe microbial pathogens from the body, large amounts of anti cific antibody reacts no more than an appropriate control bodies are also induced by intentional immunization to pro antibody would react under identical experimental condi duce specific antibodies that are used extensively in many tions. A control antibody preparation might be, for instance, biomedical and therapeutic applications. purified immunoglobulin from a pre-immune animal of the 0080 Antibody molecules are shaped somewhat like the same species, an isotype- and species-matched monoclonal letter “Y”, and consist of 4 protein chains, two heavy (H) and antibody. Tests using control antibodies to demonstrate speci two light (L) chains. Antibodies possess two distinct and ficity are recognized by one of skill in the art as appropriate spatially separate functional features. The ends of each of the and definitive. two arms of the “Y” contain the variable regions (variable 0075. In the specification and the appended claims, the heavy (V(H)) and variable light (V(L)) regions), which form singular forms include plural referents unless the context two identical antigen-binding sites. The variable regions clearly dictates otherwise. As used in this specification, the undergo a process of “affinity maturation” during the immune singular forms “a” “an and “the specifically also encom response, leading to a rapid divergence of amino acids within pass the plural forms of the terms to which they refer, unless these variable regions. The other end of the antibody mol the content clearly dictates otherwise. As used herein, unless ecule, the stem of the “Y”, contains only the two heavy specifically indicated otherwise, the word 'or' is used in the constant (CH) regions, interacts with effector cells to deter “inclusive' sense of “and/or” and not the “exclusive' sense of mine the effector functions of the antibody. There are five “eitherfor different CH region genes that encode the five different 0076. The term “about is used herein to mean approxi classes of immunoglobulins: IgM, Ig), IgG, IgA and IgE. mately, in the region of roughly, or around. When the term These constant regions, by interacting with different effector "about is used in conjunction with a numerical range, it cells and molecules, determine the immunoglobulin mol modifies that range by extending the boundaries above and ecule's biological function and biological response. below the numerical values set forth. In general, the term I0081. Each V(H) and V(L) region contains three subre “about is used herein to modify a numerical value above and gions called complementarity determining regions. These below the stated value by a variance of 20%. include CDR1-3 of the V(H) domain and CDR1-3 of the V(L) 0077. As used herein, the recitation of a numerical range domain. These six CDRS generally form the antigen binding for a variable is intended to convey that the invention may be Surface, and include those residues that hypermutate during practiced with the variable equal to any of the values within the affinity maturation phase of the immune response. The that range. Thus, for a variable that is inherently discrete, the CDR3 of the V(H) domain seems to play a dominant role in variable can be equal to any integer value of the numerical generating diversity oof both the B cell antigen receptor range, including the end-points of the range. Similarly, for a (BCR) and the T cell antigen receptor systems (Xu et al., variable that is inherently continuous, the variable can be Immunity 13:37-45 (2000)). equal to any real value of the numerical range, including the I0082. The term “antibody” or “antibodies” refers to all end-points of the range. As an example, a variable that is classes of polyclonal or monoclonal immunoglobulins, US 2010/0159477 A1 Jun. 24, 2010 including IgG, IgM, IgA, Ig|D, and IgE, including whole I0087) “Protein' is used interchangeably with polypeptide, antibodies and any antigen binding fragment thereof. This and includes protein fragments and domains as well as whole includes any combination of immunoglobulin domains or protein. chains that contains a variable region (V(H) or V(L)) that I0088 “Phosphorylatable amino acid' means any amino retains the ability to bind the immunogen. Such fragments acid that is capable of being modified by addition of a phos include F(ab) fragments (V(H)-C(H1), V(L)-C(L)); phate group, and includes both forms of Such amino acid. monovalent Fab fragments (V(H)-C(H1), V(L)-C(L)). Fv I0089. “Phosphorylatable peptide sequence” means a pep fragment (V(H)-V(L); single-chain Fv fragments (Kobayashi tide sequence comprising a phosphorylatable amino acid. et al., Steroids July; 67(8):733-42 (2002). 0090 “Phosphorylation site-specific antibody' means an 0.083 Monoclonal antibodies refer to clonal antibodies antibody that specifically binds a phosphorylatable peptide produced from fusions between immunized murine, rabbit, sequence/epitope only when phosphorylated, or only when human, or other vertebrate species, and produced by classical not phosphorylated, respectively. The term is used inter fusion technology Kohler G. Milstein C. Continuous cultures changeably with “phospho-specific' antibody. of fused cells secreting antibody of predefined specificity. 0091 Technical and scientific terms used herein have the Nature 1975 Aug. 7:256(55.17):495-7 or by alternative meth meaning commonly understood by one of skill in the art to ods which may isolate clones of immunoglobulin secreting which the present invention pertains, unless otherwise cells from transformed plasma cells. defined. Reference is made herein to various methodologies 0084. When used with respect to an antibody's binding to and materials known to those of skill in the art. Standard one phospho-form of a sequence, the expression “does not reference works setting forth the general principles of recom bind’ means that a phospho-specific antibody either does not binant DNA technology include Sambrook et al., Molecular apparently bind to the non-phospho form of the antigen as Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor ascertained in commonly used experimental detection sys Laboratory Press, New York (1989); Kaufman et al., Eds. tems (Western blotting, IHC. Immunofluorescence, etc.). Handbook of Molecular and Cellular Methods in Biology in One of skill in the art will appreciate that the expression may Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., be applicable in those instances when (1) a phospho-specific Directed Mutagenesis: A Practical Approach, IRL Press, antibody either does not apparently bind to the non-phospho Oxford (1991). Standard reference works setting forth the form of the antigen as ascertained in commonly used experi general principles of pharmacology include Goodman and mental detection systems (Western blotting, IHC. Immunof Gilman's The Pharmacological Basis of Therapeutics, 11th luorescence, etc.); (2) where there is some reactivity with the Ed., McGraw Hill Companies Inc., New York (2006). Surrounding amino acid sequence, but that the phosphory 0092 A. Identification of Phosphorylation Sites. The tar lated residue is an immunodominant feature of the reaction. get signaling protein/polypeptide phosphorylation sites dis In cases Such as these, there is an apparent difference in closed herein and listed in Table 1/FIG. 2 were discovered by affinities for the two sequences. Dilutional analyses of Such employing the modified peptide isolation and characteriza antibodies indicates that the antibodies apparent affinity for tion techniques described in U.S. Pat. No. 7,198.896 using the phosphorylated form is at least 10-100 fold higher than for cellular extracts from the following human cancer cell lines, the non-phosphorylated form; or where (3) the phospho-spe tissues and patient samples: 0 1364548-cll, 223-CLL, 293T. cific antibody reacts no more than an appropriate control 3T3 TrkB, 3T3-Src, 3T3-TrkA, 3T3-wit, 577, A172, AML antibody would react under identical experimental condi 4833, AML-6246, AML-6735, AML-7592, BaF3-10ZF, tions. A control antibody preparation might be, for instance, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), BaF3-FLT3 purified immunoglobulin from a pre-immune animal of the (D842Y), BaF3-FLT3(K663Q), BaF3-FLT3(WT), BaF3 same species, an isotype- and species-matched monoclonal FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/FGFR3, antibody. Tests using control antibodies to demonstrate speci Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P. Baf3/ ficity are recognized by one of skill in the art as appropriate Jak2(IL-3 dep), Baf3/M35 IT, Baf3/T315I, Baf3/TpoR, Baf3/ and definitive. TpoR-Y98F, Baf3/Tyk2, Baf3/V617F-jak2 (IL-3), Baf3/ 0085. “Target signaling protein/polypeptide' means any Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, CMK, protein (or polypeptide derived therefrom) enumerated in CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, EOL Column A of Table 1/FIG. 2, which is disclosed herein as 1, GDM-1, H1703, H1734, H1793, H1869, H1944, H1993, being phosphorylated in one or more cell line(s). Target sig H2023, H226, H3255, H358, H520, H82, H838, HCC1428, naling protein(s)/polypeptide(s) may be tyrosine kinases, HCC1435, HCC1806, HCC1937, HCC366, HCC827, such as TTN or BCR, or serine/threonine kinases, or direct HCT116, HEL, HL107B, HL117B, HL131A, HL131B, Substrates of Such kinases, or may be indirect Substrates HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, downstream of Such kinases in signaling pathways. Target HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, signaling protein/polypeptide where elucidated in leukemia HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS 11, KMS 18, cell lines, however one of skill in the art will appreciate that a KMS27, KOFT-K1, KY821, Karpas 299, Karpas-1 106p. target signaling protein/polypeptide may also be phosphory M-07e, M01043, MO59K, MC-1 16, MCF-10A (Y561F), lated in other cell lines (non-leukemic) harboring activated MCF-10A(Y969F), MDA-MB-453, MDA-MB-468, MEC kinase activity. 2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, I0086) “Heavy-isotope labeled peptide' (used interchange Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI ably with AQUA peptide) means a peptide comprising at least ly4, OCI-ly8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, one heavy-isotope label, which is suitable for absolute quan RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, tification or detection of a protein as described in SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, WO/03016861, “Absolute Quantification of Proteins and TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, Modified Forms Thereof by Multistage Mass Spectrometry” WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, (Gygiet al.), further discussed below. gZ21, gz68, gz73, gz74, gZB1, h1144b, h1152b, lung tumor US 2010/0159477 A1 Jun. 24, 2010 26
T26, lung tumor T57, normal human lung, pancreatic gZ21, gz68, gz73, gz74, gZB1, h1144b, h1152b, lung tumor Xenograft, patient 1, rat brain, SWA80. The isolation and iden T26, lung tumor T57, normal human lung, pancreatic tification of phosphopeptides from these cell lines, using an Xenograft, patient 1, rat brain and SWA80. immobilized general phosphotyrosine-specific antibody, or 0096. As described in more detail in the Examples, lysates an antibody recognizing the phosphorylated motif PXpSP is were prepared from these cells and digested with trypsin after described in detail in Example 1 below. In addition to the treatment with DTT and iodoacetamide to redue and alkylate protein phosphorylation sites (tyrosine) described herein, cysteine residues. Before the immunoaffinity step, peptides many known phosphorylation sites were also identified (not were pre-fractionated by reversed-phase solid phase extrac described herein). The immunoaffinity/mass spectrometric tion using Sep-Pak Cs columns to separate peptides from technique described in the 896 Patent (the “IAP' method)– other cellular components. The solid phase extraction car and employed as described in detail in the Examples—is tridges were eluted with varying steps of acetonitrile. Each briefly summarized below. lyophilized peptide fraction was redissolved in MOPS IP 0093. The IAP method employed generally comprises the buffer and treated with phosphotyrosine (P-Tyr-100, CST following steps: (a) a proteinaceous preparation (e.g. a #9411) immobilized on protein G-Sepharose. Immunoaffin digested cell extract) comprising phosphopeptides from two ity-purified peptides were eluted with 0.1%TFA and a portion or more different proteins is obtained from an organism; (b) of this fraction was concentrated with Stage or Zip tips and the preparation is contacted with at least one immobilized analyzed by LC-MS/MS, using either a LCQ or Ther general phosphotyrosine-specific antibody; (c) at least one moFinnigan LTQ ion trap mass spectrometer. Peptides were phosphopeptide specifically bound by the immobilized anti eluted from a 10 cmx75 um reversed-phase column with a body in step (b) is isolated; and (d) the modified peptide 45-min linear gradient of acetonitrile. MS/MS spectra were isolated in step (c) is characterized by mass spectrometry evaluated using the program Sequest with the NCBI human (MS) and/or tandem mass spectrometry (MS-MS). Subse protein database. quently, (e) a search program (e.g., Sequest) may be utilized 0097. This revealed the tyrosine phosphorylation sites in to substantially match the spectra obtained for the isolated, signaling pathways affected by kinase activation or active in modified peptide during the characterization of step (d) with leukemia cells. The identified phosphorylation sites and their the spectra for a known peptide sequence. A quantification parent proteins are enumerated in Table 1/FIG. 2. The step employing, e.g., SILAC or AQUA, may also be tyrosine at which phosphorylation occurs is provided in Col employed to quantify isolated peptides in order to compare umnD, and the peptide sequence encompassing the phospho peptide levels in a sample to a baseline. rylatable tyrosine residue at the site is provided in Column E. 0094. In the IAP method as employed herein, a general If a phosphorylated tyrosine was found in mouse, the ortholo phosphotyrosine-specific monoclonal antibody (commer gous site in human was identified using either Homologene or cially available from Cell Signaling Technology, Inc., Bev BLAST at NCBI; the sequence reported in column E is the erly, Mass., Cat. #9411 (p-Tyr-100)) was used in the immu phosphorylation site flanked by 7 amino acids on each side. noaffinity step to isolate the widest possible number of FIG. 2 also shows the particular type of leukemic disease (see phospho-tyrosine containing peptides from the cell extracts. Column G) and cell line(s) (see Column F) in which a par 0095 Extracts from the following human cancer cell lines, ticular phosphorylation site was discovered. tissues and patient samples were employed: 013.64548-cll, 0098. As a result of the discovery of these phosphorylation 223-CLL, 293T, 3T3 TrkB, 3T3-Src, 3T3-Trk A, 3T3-wt, sites, phospho-specific antibodies and AQUA peptides for the 577, A172, AML-4833, AML-6246, AML-6735, AML-7592, detection of and quantification of these sites and their parent BaF3-10ZF, BaF3-4ZF, BaF3-APR, BaF3-FLT3(D842V), proteins may now be produced by standard methods, as BaF3-FLT3(D842Y), BaF3-FLT3(K663Q), BaF3-FLT3 described below. These new reagents will prove highly useful (WT), BaF3-FLT3/ITD, BaF3-PRTK, BaF3-TDII, BaF3-Tel/ in, e.g., studying the signaling pathways and events underly FGFR3, Baf3, Baf3-V617F-jak2, Baf3/E255K, Baf3/H396P. ing the progression of leukemias and the identification of new Baf3/Jak2(IL-3 dep), Baf3/M351T, Baf3/T315I, Baf3/TpoR, biomarkers and targets for diagnosis and treatment of Such Baf3/TpoR-Y98F, Baf3/Tyk2, Baf3/V617F-jak2 (IL-3), diseases in a mammal. Baf3/Y253F, Baf3/cc-TpoR-IV, Baf3/p210wt, CHRF, CI-1, 0099. The methods of the present invention are intended CMK, CTV-1, DMS 53, DND41, DU-528, DU145, ELF-153, for use with any mammal that may experience the benefits of EOL-1, GDM-1, H1703, H1734, H1793, H1869, H1944, the methods of the invention. Foremost among Such mam H1993, H2023, H226, H3255, H358, H520, H82, H838, mals are humans, although the invention is not intended to be HCC1428, HCC1435, HCC1806, HCC1937, HCC366, so limited, and is applicable to veterinary uses. Thus, in HCC827, HCT116, HEL, HL107B, HL117B, HL131A, accordance with the invention, “mammals' or “mammal in HL131B, HL133A, HL53B, HL59b, HL60, HL61a, HL61b, need” include humans as well as non-human mammals, par HL66B, HL68A, HL75A, HL84A, HL97B, HL98A, HT29, ticularly domesticated animals including, without limitation, HU-3, HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS11, cats, dogs, and horses. KMS18, KMS27, KOPT-K1, KY821, Karpas 299, Karpas 0100 B. Antibodies and Cell Lines. Isolated phosphory 1106p, M-07e, M01043, MO59K, MC-1 16, MCF-10A lation site-specific antibodies that specifically bind a target (Y561F), MCF-10A(Y969F), MDA-MB-453, MDA-MB signaling protein/polypeptide disclosed in Column A of Table 468, MEC-2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, 1 only when phosphorylated (or only when not phosphory Me-F2, Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, lated) at the corresponding amino acid and phosphorylation OCI-ly4, OCI-1y8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC site listed in Columns D and E of Table 1/FIG. 2 may be K8, RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, produced by Standard antibody production methods, such as SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, anti-peptide antibody methods, using the phosphorylation TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, site sequence information provided in Column E of Table 1. WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, The TAGLN3 cytoskeletal protein phosphorylation site (ty US 2010/0159477 A1 Jun. 24, 2010 27 rosine 192) (see Row 66 of Table 1/FIG. 2) is presently diagnostic assay methods provided by the invention. For disclosed. Thus, an antibody that specifically binds this novel example, a solution containing the appropriate antigen may TAGLN3 cytoskeletal site can now be produced, e.g. by be injected into a mouse or other species and, after a sufficient immunizing an animal with a peptide antigen comprising all time (in keeping with conventional techniques), the animal is or part of the amino acid sequence encompassing the respec sacrificed and spleen cells obtained. The spleen cells are then tive phosphorylated residue (e.g., a peptide antigen compris immortalized by fusing them with myeloma cells, typically in ing the sequence set forth in Row 66, Column E, of Table 1, the presence of polyethylene glycol, to produce hybridoma SEQID NO: 65, respectively) (which encompasses the phos cells. Rabbit fusion hybridomas, for example, may be pro phorylated tyrosine at position 192 in TAGLN3, to produce an duced as described in U.S. Pat. No. 5,675,063. The hybri antibody that only binds TAGLN3 cytoskeletal protein when doma cells are then grown in a suitable selection media, Such phosphorylated at that site. as hypoxanthine-aminopterin-thymidine (HAT), and the 0101 Polyclonal antibodies of the invention may be pro duced according to standard techniques by immunizing a Supernatant Screened for monoclonal antibodies having the Suitable animal (e.g., rabbit, goat, etc.) with a peptide antigen desired specificity, as described below. The secreted antibody corresponding to the phosphorylation site of interest (i.e., a may be recovered from tissue culture Supernatant by conven phosphorylation site enumerated in Column E of Table 1, tional methods such as precipitation, ion exchange or affinity which comprises the corresponding phosphorylatable amino chromatography, or the like. acid listed in Column D of Table 1), collecting immune serum I0105 Monoclonal F fragments may also be produced in from the animal, and separating the polyclonal antibodies Escherichia coli by recombinant techniques known to those from the immune serum, in accordance with known proce skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 dures. For example, a peptide antigen corresponding to all or (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 part of the novel TES cytoskeletal phosphorylation site dis (1990). If monoclonal antibodies of one isotype are prefer closed herein (SEQID NO: 71=RTQYSCyCCK, encompass able for a particular application, particular isotypes can be ing phosphorylated tyrosine 237 (see Row 72 of Table 1)) prepared directly, by selecting from the initial fusion, or pre may be employed to produce antibodies that only bind TES pared secondarily, from a parental hybridoma secreting a when phosphorylated at Tyr 237. Similarly, a peptide com monoclonal antibody of different isotype by using the sib prising all or part of any one of the phosphorylation site selection technique to isolate class-switch variants sequences provided in Column E of Table 1 may employed as (Steplewski, et al., Proc. Natl. Acad. Sci., 82: 8653 (1985); an antigen to produce an antibody that only binds the corre Spira et al., J. Immunol. Methods, 74: 307 (1984)). sponding protein listed in Column A of Table 1 when phos 010.6 An epitope of a phosphorylation-site specific anti phorylated (or when not phosphorylated) at the correspond body of the invention is a peptide fragment consisting essen ing residue listed in Column D. If an antibody that only binds tially of about 8 to 17 amino acids including the phosphory the protein when phosphorylated at the disclosed site is latable tyrosine, wherein about 3 to 8 amino acids are desired, the peptide antigen includes the phosphorylated form positioned on each side of the phosphorylatable tyrosine (for of the amino acid. Conversely, if an antibody that only binds example, the WDR1 tyrosine 98 phosphorylation site the protein when not phosphorylated at the disclosed site is sequence disclosed in Row 83, Column E of Table 1), and desired, the peptide antigen includes the non-phosphorylated antibodies of the invention thus specifically bind a target form of the amino acid. signal protein/polypepetide comprising Such epitopic 0102 Peptide antigens suitable for producing antibodies sequence. Epitopes bound by the antibodies of the invention of the invention may be designed, constructed and employed comprise all or part of a phosphorylatable site sequence listed in accordance with well-known techniques. See, e.g., ANTI in Column E of Table 1, including the phosphorylatable BODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Har amino acid. low & Lane Eds. Cold Spring Harbor Laboratory (1988); 0107 Included in the scope of the invention are equivalent Czernik, Methods In Enzymology, 201: 264-283 (1991); Mer non-antibody molecules, such as protein binding domains or rifield, J. Am. Chem. Soc. 85: 21-49 (1962)). nucleic acid aptamers, which bind, in a phospho-specific 0103. It will be appreciated by those of skill in the art that manner, to essentially the same phosphorylatable epitope to longer or shorter phosphopeptide antigens may be employed. which the phospho-specific antibodies of the invention bind. See Id. For example, a peptide antigen may comprise the full See, e.g., Neuberger et al., Nature 312: 604 (1984). Such sequence disclosed in Column E of Table 1/FIG. 2, or it may equivalent non-antibody reagents may be suitably employed comprise additional amino acids flanking such disclosed in the methods of the invention further described below. sequence, or may comprise of only a portion of the disclosed 0.108 Antibodies provided by the invention may be any sequence immediately flanking the phosphorylatable amino type of immunoglobulins, including IgG, IgM, IgA, Ig), and acid (indicated in Column E by lowercase “y”). Typically, a IgE, including F, or antigen-recognition fragments thereof. desirable peptide antigen will comprise four or more amino The antibodies may be monoclonal or polyclonal and may be acids flanking each side of the phosphorylatable amino acid of any species of origin, including (for example) mouse, rat, and encompassing it. Polyclonal antibodies produced as rabbit, horse, or human, or may be chimeric antibodies. See, described herein may be screened as further described below. e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); 0104 Monoclonal antibodies of the invention may be pro Morrision et al., Proc. Natl. Acad. Sci. 81: 6851 (1984): duced in a hybridoma cell line according to the well-known Neuberger et al., Nature 312: 604 (1984)). The antibodies technique of Kohler and Milstein. See Nature 265: 495-97 may be recombinant monoclonal antibodies produced (1975); Kohler and Milstein, Eur: J. Immunol. 6: 511 (1976): according to the methods disclosed in U.S. Pat. No. 4,474,893 see also, Current Protocols in Molecular Biology, Ausubel et or U.S. Pat. No. 4,816,567. The antibodies may also be al. Eds. (1989). Monoclonal antibodies so produced are chemically constructed by specific antibodies made accord highly specific, and improve the selectivity and specificity of ing to the method disclosed in U.S. Pat. No. 4,676.980. US 2010/0159477 A1 Jun. 24, 2010 28
0109 The invention also provides immortalized cell lines phosphorylated (or only when not phosphorylated, as the case that produce an antibody of the invention. For example, hybri may be) at the site disclosed in corresponding Columns D/E, doma clones, constructed as described above, that produce and do not (substantially) bind to the other form (as compared monoclonal antibodies to the protein phosphorylation sites to the form for which the antibody is specific). disclosed herein are also provided. Similarly, the invention includes recombinant cells producing an antibody of the 0114 Antibodies may be further characterized via immu invention, which cells may be constructed by well known nohistochemical (IHC) staining using normal and diseased techniques; for example the antigen combining site of the tissues to evaluate phosphorylation and activation status in monoclonal antibody can be cloned by PCR and single-chain diseased tissue. IHC may be carried out according to well antibodies produced as phage-displayed recombinant anti known techniques. See, e.g., ANTIBODIES: A LABORATORY bodies or soluble antibodies in E. coli (see, e.g., ANTIBODY MANUAL, Chapter 10, Harlow & Lane Eds. Cold Spring ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul Harbor Laboratory (1988). Briefly, paraffin-embedded tissue editor.) (e.g., tumor tissue) is prepared for immunohistochemical 0110 Phosphorylation site-specific antibodies of the staining by deparaffinizing tissue sections with Xylene fol invention, whether polyclonal or monoclonal, may be lowed by ethanol; hydrating in water then PBS; unmasking screened for epitope and phospho-specificity according to antigen by heating slide in Sodium citrate buffer, incubating standard techniques. See, e.g. Czernik et al., Methods in sections in hydrogen peroxide; blocking in blocking solution; Enzymology, 201: 264-283 (1991). For example, the antibod incubating slide in primary antibody and secondary antibody; ies may be screened against the phospho and non-phospho and finally detecting using ABC avidin/biotin method accord peptide library by ELISA to ensure specificity for both the ing to manufacturer's instructions. desired antigen (i.e. that epitope including a phosphorylation 0115 Antibodies may be further characterized by flow site sequence enumerated in Column E of Table 1) and for cytometry carried out according to standard methods. See reactivity only with the phosphorylated (or non-phosphory Chow et al., Cytometry (Communications in Clinical Cytom lated) form of the antigen. Peptide competition assays may be etry) 46: 72-78 (2001). Briefly and by way of example, the carried out to confirm lack of reactivity with other phospho following protocol for cytometric analysis may be employed: epitopes on the given target signal protein/polypepetide. The samples may be centrifuged on Ficoll gradients to remove antibodies may also be tested by Western blotting against cell erythrocytes, and cells may then be fixed with 2% paraform preparations containing the signaling protein, e.g. cell lines aldehyde for 10 minutes at 37°C. followed by permeabiliza over-expressing the target protein, to confirm reactivity with tion in 90% methanol for 30 minutes on ice. Cells may then be the desired phosphorylated epitope/target. stained with the primary phosphorylation-site specific anti 0111. In an exemplary embodiment, phage display librar body of the invention (which detects a target signal protein/ ies containing more than 10" phage clones are used for high polypepetide enumerated in Table 1), washed and labeled throughput production of monoclonal antibodies that target with a fluorescent-labeled secondary antibody. Additional post-translational modification sites (e.g., phosphorylation fluorochrome-conjugated marker antibodies (e.g., CD45. sites) and, for validation and quality control, high-throughput CD34) may also be added at this time to aid in the subsequent immunohistochemistry is utilized to screen the efficacy of identification of specific hematopoietic cell types. The cells these antibodies. Western blots, protein microarrays and flow would then be analyzed on a flow cytometer (e.g., a Beckman cytometry can also be used in high-throughput screening of Coulter FC500) according to the specific protocols of the phosphorylation site-specific polyclonal or monoclonal anti instrument used. bodies of the present invention. See, e.g., Blow N. Nature, 0116. Antibodies of the invention may also be advanta 447: 741-743 (2007). geously conjugated to fluorescent dyes (e.g., Alexa488, PE) 0112 Specificity against the desired phosphorylated for use in multi-parametric analyses along with other signal epitope may also be examined by constructing mutants lack transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell ing phosphorylatable residues at positions outside the desired marker (CD34) antibodies. epitope that are known to be phosphorylated, or by mutating 0117 Phosphorylation-site specific antibodies of the the desired phospho-epitope and confirming lack of reactiv invention specifically bind to a target signaling protein/ ity. Phosphorylation-site specific antibodies of the invention polypeptide only when phosphorylated at a disclosed site, but may exhibit some limited cross-reactivity to related epitopes are not limited only to binding the human species, perse. The in non-target proteins. This is not unexpected as most anti invention includes antibodies that also bind conserved and bodies exhibit some degree of cross-reactivity, and anti-pep highly homologous or identical phosphorylation sites in tide antibodies will often cross-react with epitopes having respective Target signaling protein/polypeptide from other high homology to the immunizing peptide. See, e.g., Czernik, species (e.g., mouse, rat, monkey, yeast), in addition to bind Supra. Cross-reactivity with non-target proteins is readily ing the human phosphorylation site. Highly homologous or characterized by Western blotting alongside markers of identical sites conserved in other species can readily be iden known molecular weight. Amino acid sequences of cross tified by Standard sequence comparisons, such as using reacting proteins may be examined to identify sites highly BLAST, with the human Target signaling protein/polypeptide homologous to the Target signaling protein/polypeptide phosphorylation sites disclosed herein. epitope for which the antibody of the invention is specific. 0118 C. Heavy-Isotope Labeled Peptides (AQUA Pep 0113. In certain cases, polyclonal antisera may exhibit tides). The phosphorylation sites disclosed herein now enable Some undesirable general cross-reactivity to phosphotyrosine the production of corresponding heavy-isotope labeled pep or phosphoserine itself, which may be removed by further tides for the absolute quantification of such signaling proteins purification of antisera, e.g., over a phosphotyramine column. (both phosphorylated and not phosphorylated at a disclosed Antibodies of the invention specifically bind their target pro site) in biological samples. The production and use of AQUA tein (i.e., a protein listed in Column A of Table 1) only when peptides for the absolute quantification of proteins (AQUA) US 2010/0159477 A1 Jun. 24, 2010 29 in complex mixtures has been described. See WO/03016861, trifugation), and variability during introduction into the LC Gerber et al., Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 MS system do not affect the determined ratio of native and (2003). AQUA peptide abundances. 0119 The AQUA methodology employs the introduction I0122) An AQUA peptide standard is developed for a of a known quantity of at least one heavy-isotope labeled known phosphorylation site sequence previously identified peptide standard (which has a unique signature detectable by by the IAP-LC-MS/MS method within a target protein. One LC-SRM chromatography) into a digested biological sample AQUA peptide incorporating the phosphorylated form of the in order to determine, by comparison to the peptide standard, particular residue within the site may be developed, and a the absolute quantity of a peptide with the same sequence and second AQUA peptide incorporating the non-phosphorylated protein modification in the biological sample. Briefly, the AQUA methodology has two stages: peptide internal stan form of the residue developed. In this way, the two standards dard selection and validation and method development; and may be used to detect and quantify both the phosphorylated implementation using validated peptide internal standards to and non-phosphorylated forms of the site in a biological detect and quantify a target protein in sample. The method is sample. a powerful technique for detecting and quantifying a given I0123 Peptide internal standards may also be generated by peptide?protein within a complex biological mixture, Such as examining the primary amino acid sequence of a protein and a cell lysate, and may be employed, e.g., to quantify change in determining the boundaries of peptides produced by protease protein phosphorylation as a result of drug treatment, or to cleavage. Alternatively, a protein may actually be digested quantify differences in the level of a protein in different with a protease and a particular peptide fragment produced biological states. can then sequenced. Suitable proteases include, but are not 0120 Generally, to develop a suitable internal standard, a limited to, serine proteases (e.g., trypsin, hepsin), metallo particular peptide (or modified peptide) within a target pro proteases (e.g., PUMP1), chymotrypsin, cathepsin, pepsin, tein sequence is chosen based on its amino acid sequence and thermolysin, carboxypeptidases, etc. the particular protease to be used to digest. The peptide is then 0.124. A peptide sequence within a target protein is generated by Solid-phase peptide synthesis such that one resi selected according to one or more criteria to optimize the use due is replaced with that same residue containing stable iso of the peptide as an internal standard. Preferably, the size of topes (C, 'N). The result is a peptide that is chemically the peptide is selected to minimize the chances that the pep identical to its native counterpartformed by proteolysis, but is tide sequence will be repeated elsewhere in other non-target easily distinguishable by MS via a 7-Damass shift. A newly proteins. Thus, a peptide is preferably at least about 6 amino synthesized AQUA internal standard peptide is thenevaluated acids. The size of the peptide is also optimized to maximize by LCMS/MS. This process provides qualitative information ionization frequency. A workable range is about 7 to 15 amino about peptide retention by reverse-phase chromatography, acids. A peptide sequence is also selected that is not likely to ionization efficiency, and fragmentation via collision-in be chemically reactive during mass spectrometry, thus duced dissociation. Informative and abundant fragment ions sequences comprising cysteine, tryptophan, or methionine for sets of native and internal standard peptides are chosen are avoided. and then specifically monitored in rapid succession as a func 0.125. A peptide sequence that does not include a modified tion of chromatographic retention to form a selected reaction region of the target region may be selected so that the peptide monitoring (LC-SRM) method based on the unique profile of internal standard can be used to determine the quantity of all the peptide standard. forms of the protein. Alternatively, a peptide internal standard 0121 The second stage of the AQUA strategy is its imple encompassing a modified amino acid may be desirable to mentation to measure the amount of a protein or modified detect and quantify only the modified form of the target protein from complex mixtures. Whole cell lysates are typi protein. Peptide standards for both modified and unmodified cally fractionated by SDS-PAGE gel electrophoresis, and regions can be used together, to determine the extent of a regions of the gel consistent with protein migration are modification in a particular sample (i.e. to determine what excised. This process is followed by in-gel proteolysis in the fraction of the total amount of protein is represented by the presence of the AQUA peptides and LC-SRM analysis. (See modified form). For example, peptide standards for both the Gerber et al., supra.) AQUA peptides are spiked in to the phosphorylated and unphosphorylated form of a protein complex peptide mixture obtained by digestion of the whole known to be phosphorylated at a particular site can be used to cell lysate with a proteolytic enzyme and Subjected to immu quantify the amount of phosphorylated form in a sample. noaffinity purification as described above. The retention time 0.126 The peptide is labeled using one or more labeled and fragmentation pattern of the native peptide formed by amino acids (i.e. the label is an actual part of the peptide) or digestion (e.g., trypsinization) is identical to that of the less preferably, labels may be attached after synthesis accord AQUA internal standard peptide determined previously; thus, ing to standard methods. Preferably, the label is a mass LC-MS/MS analysis using an SRM experiment results in the altering label selected based on the following considerations: highly specific and sensitive measurement of both internal the mass should be unique to shift fragment masses produced standard and analyte directly from extremely complex pep by MS analysis to regions of the spectrum with low back tide mixtures. Because an absolute amount of the AQUA ground; the ion mass signature component is the portion of peptide is added (e.g., 250 fmol), the ratio of the areas under the labeling moiety that preferably exhibits a unique ion mass the curve can be used to determine the precise expression signature in MS analysis; the Sum of the masses of the con levels of a protein orphosphorylated form of a protein in the stituent atoms of the label is preferably uniquely different original cell lysate. In addition, the internal standard is than the fragments of all the possible amino acids. As a result, present during in-gel digestion as native peptides are formed, the labeled amino acids and peptides are readily distinguished Such that peptide extraction efficiency from gel pieces, abso from unlabeled ones by the ion/mass pattern in the resulting lute losses during sample handling (including vacuum cen mass spectrum. Preferably, the ion mass signature component US 2010/0159477 A1 Jun. 24, 2010 30 imparts a mass to a protein fragment that does not match the the area under the curve (AUC) for both peptide standard and residue mass for any of the 20 natural amino acids. target peptide peaks are calculated. The ratio of the two areas 0127. The label should be robust under the fragmentation provides the absolute quantification that can be normalized conditions of MS and not undergo unfavorable fragmenta for the number of cells used in the analysis and the protein's tion. Labeling chemistry should be efficient under a range of molecular weight, to provide the precise number of copies of conditions, particularly denaturing conditions, and the the protein percell. Further details of the AQUA methodology labeled tag preferably remains soluble in the MS buffer sys are described in Gygiet al., and Gerber et al. Supra. tem of choice. The label preferably does not suppress the 0.132. In accordance with the present invention, AQUA ionization efficiency of the protein and is not chemically internal peptide standards (heavy-isotope labeled peptides) reactive. The label may contain a mixture of two or more may now be produced, as described above, for any of the isotopically distinct species to generate a unique mass spec phosphorylation sites disclosed herein. Peptide standards for trometric pattern at each labeled fragment position. Stable a given phosphorylation site (e.g., the tyrosine 328 in isotopes, such as H, C, N, O, O, or 'S, are sutable TOP2A—see Row 87 of Table 1) may be produced for both labels. Pairs of peptide internal standards that incorporate a the phosphorylated and non-phosphorylated forms of the site different isotope label may also be prepared. Amino acid (e.g., see PKCD site sequence in Column E. Row 123 of Table residues into which a heavy isotope label may be incorporated 1 (SEQ ID NO: 122) and such standards employed in the include leucine, proline, Valine, and phenylalanine. AQUA methodology to detect and quantify both forms of 0128 Peptide internal standards are characterized accord Such phosphorylation site in a biological sample. ing to their mass-to-charge (m/z) ratio, and preferably, also 0.133 AQUA peptides of the invention may comprise all, according to their retention time on a chromatographic col or part of a phosphorylation site peptide sequence disclosed umn (e.g. an HPLC column). Internal standards that co-elute herein (see Column E of Table 1/FIG. 2). In an embodiment, with unlabeled peptides of identical sequence are selected as an AQUA peptide of the invention comprises a phosphoryla optimal internal standards. The internal standard is then ana tion site sequence disclosed herein in Table 1/FIG. 2. For lyzed by fragmenting the peptide by any suitable means, for example, an AQUA peptide of the invention for detection/ example by collision-induced dissociation (CID) using, e.g., quantification of SMRT transcriptional regulator protein argon or helium as a collision gas. The fragments are then when phosphorylated at tyrosine Y2249 may comprise the analyzed, for example by multi-stage mass spectrometry sequence SAVyPLLYR (y-phosphotyrosine), which com (MS) to obtain a fragmention spectrum, to obtain a peptide prises phosphorylatable tyrosine 2249 (see Row 177, Column fragmentation signature. Preferably, peptide fragments have E: (SEQID NO: 176)). Heavy-isotope labeled equivalents of significant differences in m/z ratios to enable peaks corre the peptides enumerated in Table 1/FIG. 2 (both in phospho sponding to each fragment to be well separated, and a signa rylated and unphosphorylated form) can be readily synthe ture that is unique for the target peptide is obtained. If a sized and their unique MS and LC-SRM signature deter Suitable fragment signature is not obtained at the first stage, mined, so that the peptides are validated as AQUA peptides additional stages of MS are performed until a unique signa and ready for use in quantification experiments. ture is obtained. I0134. The phosphorylation site peptide sequences dis 0129. Fragment ions in the MS/MS and MS spectra are closed herein (see Column E of Table 1/FIG.2) are well suited typically highly specific for the peptide of interest, and, in for development of corresponding AQUA peptides, since the conjunction with LC methods, allow a highly selective means IAP method by which they were identified (see Part A above of detecting and quantifying a target peptide?protein in a and Example 1) inherently confirmed that Such peptides are in complex protein mixture. Such as a cell lysate, containing fact produced by enzymatic digestion (trypsinization) and are many thousands or tens of thousands of proteins. Any bio in fact suitably fractionated/ionized in MS/MS. Thus, heavy logical sample potentially containing a target protein/peptide isotope labeled equivalents of these peptides (both in phos of interest may be assayed. Crude or partially purified cell phorylated and unphosphorylated form) can be readily Syn extracts may be employed. Generally, the sample has at least thesized and their unique MS and LC-SRM signature 0.01 mg of protein, typically a concentration of 0.1-10 determined, so that the peptides are validated as AQUA pep mg/mL, and may be adjusted to a desired buffer concentration tides and ready for use in quantification experiments. and pH. 0.135 Accordingly, the invention provides heavy-isotope 0130. A known amount of a labeled peptide internal stan labeled peptides (AQUA peptides) for the detection and/or dard, preferably about 10 femtomoles, corresponding to a quantification of any of the phosphorylation sites disclosed in target protein to be detected/quantified is then added to a Table 1/FIG. 2 (see Column E) and/or their corresponding biological sample, such as a cell lysate. The spiked sample is parent proteins/polypeptides (see Column A). A phosphopep then digested with one or more protease(s) for a suitable time tide sequence comprising any of the phosphorylation period to allow digestion. A separation is then performed sequences listed in Table 1 may be considered an AQUA (e.g., by HPLC, reverse-phase HPLC, capillary electrophore peptide of the invention. For example, an AQUA peptide sis, ion exchange chromatography, etc.) to isolate the labeled comprising the sequence SAVyPLLYR (SEQ ID NO: 176) internal standard and its corresponding target peptide from (where y may be either phosphotyrosine or tyrosine, and other peptides in the sample. Microcapillary LC is a method where V-labeled valine (e.g., ''C)) is provided for the quan contemplated. tification of phosphorylated (or non-phosphorylated) diapha 0131 Each isolated peptide is then examined by monitor nous (Tyr2249) in a biological sample (see Row 177 of Table ing of a selected reaction in the MS. This involves using the 1, tyrosine 2249 being the phosphorylatable residue within prior knowledge gained by the characterization of the peptide the site). It will be appreciated that a larger AQUA peptide internal standard and then requiring the MS to continuously comprising a disclosed phosphorylation site sequence (and monitor a specific ion in the MS/MS or MS" spectrum for both additional residues downstream or upstream of it) may also be the peptide of interest and the internal standard. After elution, constructed. Similarly, a smaller AQUA peptide comprising US 2010/0159477 A1 Jun. 24, 2010
less than all of the residues of a disclosed phosphorylation site detectable signal. Similar specimens as described above may sequence (but still comprising the phosphorylatable residue be used. The antibody is generally immobilized on a Support, enumerated in ColumnD of Table 1/FIG.2) may alternatively Such as a bead, plate or slide, and contacted with the specimen be constructed. Such larger or shorter AQUA peptides are Suspected of containing the antigen in a liquid phase. The within the scope of the present invention, and the selection Support is then separated from the liquid phase and either the and production of AQUA peptides may be carried out as Support phase or the liquid phase is examined for a detectable described above (see Gygiet al., Gerber et al., Supra.). signal employing means for producing Such signal. The sig 0.136 Certain subsets of AQUA peptides provided by the nal is related to the presence of the analyte in the specimen. invention are described above (corresponding to particular Means for producing a detectable signal include the use of protein types/groups in Table 1, for example, tyrosine protein radioactive labels, fluorescent labels, enzyme labels, and so kinases or adaptor/scaffold proteins). Example 4 is provided forth. For example, if the antigen to be detected contains a to further illustrate the construction and use, by standard second binding site, an antibody which binds to that site can methods described above, of exemplary AQUA peptides pro be conjugated to a detectable group and added to the liquid vided by the invention. For example, the above-described phase reaction solution before the separation step. The pres AQUA peptides corresponding to both the phosphorylated ence of the detectable group on the solid Support indicates the and non-phosphorylated forms of the disclosed Tel transcrip presence of the antigen in the test sample. Examples of Suit tional regulator protein tyrosine 402 phosphorylation site (see able immunoassays are the radioimmunoassay, immunofluo Row 213 of Table 1/FIG. 2) may be used to quantify the rescence methods, enzyme-linked immunoassays, and the amount of phosphorylated Tel (Tyr 402) in a biological like. sample, e.g., a tumor cell sample (or a sample before or after 0.141. Immunoassay formats and variations thereof that treatment with a test drug). may be useful for carrying out the methods disclosed herein 0.137 AQUA peptides of the invention may also be are well known in the art. Seegenerally E. Maggio, Enzyme employed within a kit that comprises one or multiple AQUA Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); peptide(s) provided herein (for the quantification of a Target see also, e.g., U.S. Pat. No. 4,727,022; U.S. Pat. No. 4,659, signaling protein/polypeptide disclosed in Table 1/FIG. 2), 678; U.S. Pat. No. 4,376,110. Conditions suitable for the and, optionally, a second detecting reagent conjugated to a formation of reagent-antibody complexes are well described. detectable group. For example, a kit may include AQUA Seeid. Monoclonal antibodies of the invention may be used in peptides for both the phosphorylated and non-phosphory a “two-site' or “sandwich' assay, with a single cell line serv lated form of a phosphorylation site disclosed herein. The ing as a source for both the labeled monoclonal antibody and reagents may also include ancillary agents such as buffering the bound monoclonal antibody. Such assays are described in agents and protein stabilizing agents, e.g., polysaccharides U.S. Pat. No. 4,376, 110. The concentration of detectable and the like. The kit may further include, where necessary, reagent should be sufficient Such that the binding of a Target other members of the signal-producing system of which sys signaling protein/polypeptide is detectable compared to tem the detectable group is a member (e.g., enzyme Sub background. strates), agents for reducing background interference in a test, 0.142 Phosphorylation site-specific antibodies disclosed control reagents, apparatus for conducting a test, and the like. herein may be conjugated to a solid Support Suitable for a The test kit may be packaged in any suitable manner, typically diagnostic assay (e.g., beads, plates, slides or wells formed with all elements in a single container along with a sheet of from materials such as latex or polystyrene) in accordance printed instructions for carrying out the test. with known techniques, such as precipitation. Antibodies, or 0138 AQUA peptides provided by the invention will be other target protein or target site-binding reagents, may like useful in the further study of signal transduction anomalies wise be conjugated to detectable groups such as radiolabels associated with diseases Such as for example cancer, includ (e.g., S.I. ''I), enzyme labels (e.g., horseradish peroxi ing leukemias, and in identifying diagnostic/bio-markers of dase, alkaline phosphatase), and fluorescent labels (e.g., fluo these diseases, new potential drug targets, and/or in monitor rescein) in accordance with known techniques. ing the effects of test compounds on Target Signaling Pro 0.143 Antibodies of the invention may also be optimized teins/Polypeptides and pathways. for use in a flow cytometry (FC) assay to determine the 0.139. D. Immunoassay Formats. Antibodies provided by activation/phosphorylation status of a Target signaling pro the invention may be advantageously employed in a variety of tein/polypeptide in patients before, during, and after treat standard immunological assays (the use of AQUA peptides ment with a drug targeted at inhibiting phosphorylation of provided by the invention is described separately above). Such a protein at the phosphorylation site disclosed herein. Assays may be homogeneous assays or heterogeneous For example, bone marrow cells or peripheral blood cells assays. In a homogeneous assay the immunological reaction from patients may be analyzed by flow cytometry for Target usually involves a phosphorylation-site specific antibody of signaling protein/polypeptide phosphorylation, as well as for the invention), a labeled analyte, and the sample of interest. markers identifying various hematopoietic cell types. In this The signal arising from the label is modified, directly or manner, activation status of the malignant cells may be spe indirectly, upon the binding of the antibody to the labeled cifically characterized. Flow cytometry may be carried out analyte. Both the immunological reaction and detection of the according to standard methods. See, e.g. Chow et al., Cytom extent thereof are carried out in a homogeneous solution. etry (Communications in Clinical Cytometry) 46: 72-78 Immunochemical labels that may be employed include free (2001). Briefly and by way of example, the following proto radicals, radioisotopes, fluorescent dyes, enzymes, bacte col for cytometric analysis may be employed: fixation of the riophages, coenzymes, and so forth. cells with 1% para-formaldehyde for 10 minutes at 37° C. 0140. In a heterogeneous assay approach, the reagents are followed by permeabilization in 90% methanol for 30 min usually the specimen, a phosphorylation-site specific anti utes on ice. Cells may then be stained with the primary anti body of the invention, and Suitable means for producing a body (a phospho-specific antibody of the invention), washed US 2010/0159477 A1 Jun. 24, 2010 32 and labeled with a fluorescent-labeled secondary antibody. to cover alternatives, modifications, and equivalents as may Alternatively, the cells may be stained with a fluorescent be included within the spirit and scope of the invention as labeled primary antibody. The cells would then be analyzed defined by the appended claims. In the description, numerous on a flow cytometer (e.g., a Beckman Coulter EPICS-XL) specific details are set forth in order to provide a thorough according to the specific protocols of the instrument used. understanding of the present invention. The present invention Such an analysis would identify the presence of activated may be practiced without some or all of these specific details. Target Signaling Protein(s)/Polypeptide(s) in the malignant In other instances, well known process operations have not cells and reveal the drug response on the targeted protein. been described in detail, in order not to unnecessarily obscure 0144. Alternatively, antibodies of the invention may be the present invention. employed in immunohistochemical (IHC) staining to detect 0.148. The following examples are intended to further differences in signal transduction or protein activity using illustrate certain embodiments of the invention and are not normal and diseased tissues. IHC may be carried out accord limiting in nature. Those skilled in the art will recognize, or be ing to well-known techniques. See, e.g., Antibodies. A Labo able to ascertain, using no more than routine experimentation, ratory Manual, Supra. Briefly, paraffin-embedded tissue (e.g., numerous equivalents to the specific Substances and proce tumor tissue) is prepared for immunohistochemical staining dures described herein. by deparaffinizing tissue sections with xylene followed by 0149 Any suitable materials and/or methods known to ethanol; hydrating in water then PBS; unmasking antigen by those of skill can be utilized in carrying out the present inven heating slide in Sodium citrate buffer, incubating sections in tion. However, materials and methods are described. Materi hydrogen peroxide; blocking in blocking solution; incubating als, reagents and the like to which reference is made in the slide in primary antibody and secondary antibody; and finally following description and examples are obtainable from com detecting using ABC avidin/biotin method according to mercial Sources, unless otherwise noted. manufacturer's instructions. 0145 Antibodies of the invention may be also be opti Example 1 mized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN. Isolation of Phosphotyrosine-Containing Peptides LuminexTM and/or BioplexTM assay formats, or otherwise from Extracts of Cancer Cell Lines and Identification optimized for antibody array formats, such as reversed-phase of Phosphorylation Sites array applications (see, e.g., Paweletz et al., Oncogene 0150 IAP isolation techniques were employed to identify 20(16): 1981-89 (2001)). Accordingly, in another embodi phosphotyrosine containing peptides in cell extracts from the ment, the invention provides a method for the multiplex following human cancer cell lines, tissues and patient cell detection of phosphorylation in a biological sample, the lines: 0 1364548-cll, 223-CLL, 293T, 3T3 TrkB, 3T3-Src, method comprising utilizing two or more antibodies or 3T3-TrkA, 3T3-wit, 577, A 172, AML-4833, AML-6246, AQUA peptides of the invention to detect the presence of two AML-6735, AML-7592, BaF3-10ZF, BaF3-4ZF, BaF3 or more phosphorylated proteins enumerated in Column A of APR, BaF3-FLT3(D842V), BaF3-FLT3(D842Y), BaF3 Table 1/FIG. 2. In an embodiment, two to five antibodies or FLT3(K663Q), BaF3-FLT3(WT), BaF3-FLT3/ITD, BaF3 AQUA peptides of the invention are employed in the method. PRTK, BaF3-TDII, BaF3-Tel/FGFR3, Baf3, Baf3-V617F In another embodiment, six to ten antibodies or AQUA pep jak2, Baf3/E255K, Baf3/H396P. Baf3/Jak2(IL-3 dep), Baf3/ tides of the invention are employed, while in another embodi M351T, Baf3/T315I, Baf3/TpoR, Baf3/TpoR-Y98F, Baf3/ ment eleven to twenty Such reagents are employed. Tyk2, Baf3/V617F-jak2 (IL-3), Baf3/Y253F, Baf3/cc-TpoR 014.6 Antibodies and/or AQUA peptides of the invention IV, Baf3/p210wt, CHRF, CI-1, CMK, CTV-1, DMS 53, may also be employed within a kit that comprises at least one DND41, DU-528, DU145, ELF-153, EOL-1, GDM-1, phosphorylation site-specific antibody or AQUA peptide of H1703, H1734, H1793, H1869, H1944, H1993, H2023, the invention (which binds to or detects a Target signaling H226, H3255, H358, H520, H82, H838, HCC1428, protein/polypeptide disclosed in Table 1/FIG. 2), and, option HCC1435, HCC1806, HCC1937, HCC366, HCC827, ally, a second antibody conjugated to a detectable group. In HCT116, HEL, HL107B, HL117B, HL131A, HL131B, Some embodies, the kit is Suitable for multiplex assays and HL133A, HL53B, HL59b, HL60, HL61a, HL61b, HL66B, comprises two or more antibodies or AQUA peptides of the HL68A, HL75A, HL84A, HL97B, HL98A, HT29, HU-3, invention, and in some embodiments, comprises two to five, HUVEC, Jurkat, K562, KG-1, KG 1-A, KMS 11, KMS 18, six to ten, or eleven to twenty reagents of the invention. The KMS27, KOPT-K 1, KY821, Karpas 299, Karpas-1 106p. kit may also include ancillary agents such as buffering agents M-07e, M01043, MO59K, MC-1 16, MCF-10A (Y561F), and protein stabilizing agents, e.g., polysaccharides and the MCF-10A(Y969F), MDA-MB-453, MDA-MB-468, MEC like. The kit may further include, where necessary, other 2, MKPL-1, ML-1, MO-91, MOLT15, MV4-11, Me-F2, members of the signal-producing system of which system the Molm 14, Monomac 6, NCI-N87, Nomo-1, OCI-M1, OCI detectable group is a member (e.g., enzyme Substrates), ly4, OCI-1y8, OCI/AML2, OPM-1, PL21, Pfeiffer, RC-K8, agents for reducing background interference in a test, control RI-1, SCLC T1, SEM, SK-N-AS, SK-N-MC, SKBR3, reagents, apparatus for conducting a test, and the like. The test SR-786, SU-DHL1, SUP-M2, SUPT-13, SuDHL5, T17, kit may be packaged in any suitable manner, typically with all TRE-cll patient, TS, UT-7, VAL, Verona, Verona 1, Verona 4, elements in a single container along with a sheet of printed WSU-NHL, XG2, Z-55, cs001, cs015, cs025, cs041, cs042, instructions for carrying out the test. gZ21. g768, gz73, gz74, gZB1, h1144b, hl 152b, lung tumor 0147 Reference is made hereinafter in detail to specific T26, lung tumor T57, normal human lung, pancreatic embodiments of the invention. While the invention will be Xenograft, patient 1, rat brain and SWA80. described in conjunction with these specific embodiments, it 0151 Tryptic phosphotyrosine containing peptides were will be understood that it is not intended to limit the invention purified and analyzed from extracts of each of the cell lines to Such specific embodiments. On the contrary, it is intended mentioned above, as follows. Cells were cultured in DMEM US 2010/0159477 A1 Jun. 24, 2010
medium or RPMI 1640 medium supplemented with 10% fetal min (eluate 1), followed by a wash of the beads (eluate 2) with bovine serum and penicillin/streptomycin. 40 ul of 0.15% TFA. Both eluates were combined. 0152 Suspension cells were harvested by low speed cen trifugation. After complete aspiration of medium, cells were Analysis by LC-MS/MS Mass Spectrometry. resuspended in 1 mL lysis buffer per 1.25x10 cells (20 mM (O157 40 ul or more of IAP eluate were purified by 0.2 ul HEPES pH 8.0, 9 Murea, 1 mM sodium vanadate, supple StageTips (Proxeon, Staermosegaardsvei 6, DK-5230 mented or not with 2.5 mM sodium pyro-phosphate, 1 mM Odense M. Denmark) or ZipTips(R (produced by Millipore(R), B-glycerol-phosphate) and Sonicated. Billerica Mass.). Peptides were eluted from the microcol 0153 Sonicated cell lysates were cleared by centrifuga umns with 1 ul of 40% MeCN, 0.1%TFA (fractions I and II) tion at 20,000xg, and proteins were reduced with DTT at a or 1 ul of 60% MeCN, 0.1%TFA (fraction III) into 7.6 ul of final concentration of 4.1 mM and alkylated with iodoaceta 0.4% acetic acid/0.005% heptafluorobutyric acid. This mide at 8.3 mM. For digestion with trypsin, protein extracts sample was loaded onto a 10 cmx75um PicoFrit(R) capillary were diluted in 20 mMHEPES pH 8.0 to a final concentration column (produced by New Objective, Woburn, Mass.) packed of 2 Murea and soluble TLCKR)-trypsin (Worthington(R) with Michrom Magic Bullets(R C18 AQ reversed-phase resin (Michrom Bioresources, Auburn Calif.) using a FamosTM Biochemcial Corporation, Lakewood, N.J.) was added at autosampler with an inert sample injection valve (DioneXOR, 10-20 g/mL. Digestion was performed for 1-2 days at room Sunnyvale, Calif.). The column was then developed with a temperature. 45-minlinear gradient of acetonitrile delivered at 200 ml/min 0154 Trifluoroacetic acid (TFA) was added to protein (using an Ultimate R pump, DioneXR), Sunnyvale, Calif.), and digests to a final concentration of 1%, precipitate was tandem mass spectra were collected in a data-dependent man removed by centrifugation, and digests were loaded onto ner with an LTQR (produced by Thermo (R) Finnigan R. San, Sep-Pak R. Cls columns (provided by Waters Corporation, Jose, Calif.), ion trap mass spectrometer essentially as Milford, Mass.) equilibrated with 0.1% TFA. A column vol described by Gygiet al., Supra. ume of 0.7-1.0 ml was used per 2x10 cells. Columns were washed with 15 volumes of 0.1%TFA, followed by 4 volumes Database Analysis & Assignments. of 5% acetonitrile (MeCN) in 0.1% TFA. Peptide fraction I 0158 MS/MS spectra were evaluated using TurboSe was obtained by eluting columns with 2 volumes each of 8, questTM in the Sequest(R) (owned by Thermo (R) Finnigan R. San 12, and 15% MeCN in 0.1% TFA and combining the eluates. Jose, Calif.) Browser package (v. 27, rev. 12) supplied as part Fractions II and III were a combination of eluates after eluting of BioWorksTM 3.0 (Thermo(R) Finnigan R, San Jose, Calif.). columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, Individual MS/MS spectra were extracted from the raw data 35, 40% MeCN in 0.1%TFA, respectively. All peptide frac file using the Sequest(R) Browser program CreateDtaTM tions were lyophilized. (owned by Thermo R. Finnigan R. San Jose, Calif.), with the 0155 Peptides from each fraction corresponding to 2x10 following settings: bottom MW, 700; top MW, 4,500; mini cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HClor mum number of ions, 20; minimum TIC, 4x10; and precur 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM Sor charge state, unspecified. Spectra were extracted from the NaCl) and insoluble material was removed by centrifugation. beginning of the raw data file before sample injection to the IAP was performed on each peptide fraction separately. The end of the eluting gradient. The IonCuestTM and VulDtaTM phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Sig (owned by Thermo R. Finnigan R. San Jose, Calif.) programs naling Technology(R), Inc., Danvers, Mass. catalog number were not used to further select MS/MS spectra for Sequest(R) analysis. MS/MS spectra were evaluated with the following 9411) was coupled at 4 mg/ml beads to protein G or protein A TurboSequestTM parameters: peptide mass tolerance, 2.5; agarose (Roche?R), Basel, Switzerland), respectively. Immo fragmention tolerance, 0.0; maximum number of differential bilized antibody (15ul, 60 ug) was added as 1:1 slurry in IAP amino acids per modification, 4, mass type parent, average; buffer to 1.4 ml of each peptide fraction, and the mixture was mass type fragment, average; maximum number of internal incubated overnight at 4°C. with gentle rotation. The immo cleavage sites, 10; neutral losses of water and ammonia from bilized antibody beads were washed three times with 1 ml b and y ions were considered in the correlation analysis. IAP buffer and twice with 1 ml water, all at 4° C. Peptides Proteolytic enzyme was specified except for spectra collected were eluted from beads by incubation with 75ul of 0.1%TFA from elastase digests. at room temperature for 10 minutes. 0159. Searches were performed against the NCBI human 0156 Alternatively, one single peptide fraction was protein database (as released on Aug. 24, 2004 and containing obtained from Sep-Pak C18 columns by elution with 2 vol 27, 960 protein sequences). Cysteine carboxamidomethyla umes each of 10%, 15%, 20%, 25%, 30%, 35% and 40% tion was specified as a static modification, and phosphoryla acetonitirile in 0.1% TFA and combination of all eluates. IAP tion was allowed as a variable modification on serine, threo on this peptide fraction was performed as follows: After lyo nine, and tyrosine residues or on tyrosine residues alone. It philization, peptide was dissolved in 1.4 ml IAP buffer was determined that restricting phosphorylation to tyrosine (MOPS pH 7.2, 10 mM sodium phosphate, 50 mMNaCl) and residues had little effect on the number of phosphorylation insoluble material was removed by centrifugation. Immobi sites assigned. Furthermore, it should be noted that certain lized antibody (40 ul, 160 g) was added as 1:1 slurry in IAP peptides were originally isolated in mouse and later normal buffer, and the mixture was incubated overnight at 4°C. with ized to human sequences as shown by Table 1/FIG. 2. gentle shaking. The immobilized antibody beads were 0160. In proteomics research, it is desirable to validate washed three times with 1 ml IAP buffer and twice with 1 ml protein identifications based solely on the observation of a water, all at 4°C. Peptides were eluted from beads by incu single peptide in one experimental result, in order to indicate bation with 40 ul of 0.15% TFA at room temperature for 10 that the protein is, in fact, present in a sample. This has led to US 2010/0159477 A1 Jun. 24, 2010 34 the development of statistical methods for validating peptide constructing a synthetic peptide antigen comprising the phos assignments, which are not yet universally accepted, and phorylation site sequence and then immunizing an animal to guidelines for the publication of protein and peptide identifi raise antibodies against the antigen, as further described cation results (see Carr et al., Mol. Cell. Proteomics 3: 531 below. Production of exemplary polyclonal antibodies is pro 533 (2004)), which were followed in this Example. However, vided below. because the immunoaffinity strategy separates phosphory lated peptides from unphosphorylated peptides, observing A. VCP (Tyrosine 644). just one phosphopeptide from a protein is a common result, 0.165. A 13 amino acid phospho-peptide antigen, since many phosphorylated proteins have only one tyrosine LDKLly IPLPDEK (wherey phosphotyrosine) that corre phosphorylated site. For this reason, it is appropriate to use sponds to the sequence encompassing the tyrosine 644 phos additional criteria to validate phosphopeptide assignments. phorylation site in human VCP cell cycle regulation protein Assignments are likely to be correct if any of these additional (see Row 28 of Table 1: SEQID NO: 27), plus cysteine on the criteria are met: (i) the same sequence is assigned to co C-terminal for coupling, is constructed according to standard eluting ions with different charge states, since the MS/MS synthesis techniques using, e.g., a Rainin/Protein Technolo spectrum changes markedly with charge state; (ii) the site is gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A found in more than one peptide sequence context due to LABORATORY MANUAL, supra.; Merrifield, supra. This pep sequence overlaps from incomplete proteolysis or use of pro tide is then coupled to KLH and used to immunize animals to teases other than trypsin; (iii) the site is found in more than produce (and Subsequently screen) phospho-specific VCP one peptide sequence context due to homologous but not (tyró43) polyclonal antibodies as described in Immunization/ identical protein isoforms; (iv) the site is found in more than one peptide sequence context due to homologous but not Screening below. identical proteins among species; and (v) sites validated by MS/MS analysis of synthetic phosphopeptides correspond B. HSP90B (Tyrosine 192). ing to assigned sequences, since the ion trap mass spectrom 0166 An 16 amino acid phospho-peptide antigen, eter produces highly reproducible MS/MS spectra. The last VILHLKEDQTEy*LEER (where y-phosphotyrosine) that criterion is routinely employed to confirm novel site assign corresponds to the sequence encompassing the tyrosine 192 ments of particular interest. phosphorylation site in human HSP90B chaperone protein 0161 All spectra and all sequence assignments made by (see Row 30 of Table 1 (SEQIDNO: 29)), plus cysteine on the Sequest were imported into a relational database. The follow C-terminal for coupling, is constructed according to standard ing Sequest scoring thresholds were used to select phospho synthesis techniques using, e.g., a Rainin/Protein Technolo peptide assignments that are likely to be correct: RSp-6, gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A XCorre2.2, and DeltaCN>0.099. Further, the assigned LABORATORY MANUAL, supra.; Merrifield, supra. This pep sequences could be accepted or rejected with respect to accu tide is then coupled to KLH and used to immunize animals to racy by using the following conservative, two-step process. produce (and Subsequently screen) phospho-specific 0162. In the first step, a Subset of high-scoring sequence HSP90B (tyr 191) polyclonal antibodies as described in assignments should be selected by filtering for XCorr values Immunization/Screening below. 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 C. TSN (Tyrosine 210). this subset should be rejected if any of the following criteria 0.167 A 12 amino acid phospho-peptide antigen, were satisfied: (i) the spectrum contains at least one major KVEEVVy*DLSIR (where y-phosphotyrosine) that corre peak (at least 10% as intense as the most intense ion in the sponds to the sequence encompassing the tyrosine 210 phos spectrum) that can not be mapped to the assigned sequence as phorylation site in human catalase chromatin or DNA bind ana, b, ory ion, as anion arising from neutral-loss of water or ing/repair/replication protein (see Row 44 of Table 1 (SEQID ammonia from ab ory ion, or as a multiply protonated ion; (ii) NO: 43), plus cysteine on the C-terminal for coupling, is the spectrum does not contain a series of bory ions equivalent constructed according to standard synthesis techniques using, to at least six uninterrupted residues; or (iii) the sequence is e.g., a Rainin/Protein Technologies, Inc., Symphony peptide not observed at least five times in all the studies conducted synthesizer. See ANTIBODIES: A LABORATORY MANUAL, (except for overlapping sequences due to incomplete pro supra.; Merrifield, supra. This peptide is then coupled to KLH teolysis or use of proteases other than trypsin). and used to immunize animals to produce (and Subsequently 0163. In the second step, assignments with below-thresh screen) phospho-specific TSN (tyr 210) antibodies as old scores should be accepted if the low-scoring spectrum described in Immunization/Screening below. shows a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference Immunization/Screening. library-searching strategy. 0168 A synthetic phospho-peptide antigenas described in A-C above is coupled to KLH, and rabbits are injected intra Example 2 dermally (ID) on the back with antigen in complete Freunds Production of Phospho-Specific Polyclonal Antibod adjuvant (384 ug antigen per rabbit). The rabbits are boosted ies for the Detection of Target Signal Protein/ with same antigen in incomplete Freund adjuvant (250 ug Polypepetide Phosphorylation antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affin 0164 Polyclonal antibodies that specifically bind a target ity chromatography by standard methods (see ANTIBODIES: A signal protein/polypepetide only when phosphorylated at the LABORATORY MANUAL, Cold Spring Harbor, supra.). The respective phosphorylation site disclosed herein (see Table eluted immunoglobulins are further loaded onto a non-phos 1/FIG. 2) are produced according to standard methods by first phorylated synthetic peptide antigen-resin Knotes column to US 2010/0159477 A1 Jun. 24, 2010
pull out antibodies that bind the non-phosphorylated form of as further described below. Production of exemplary mono the phosphorylation site. The flow through fraction is col clonal antibodies is provided below. lected and applied onto a phospho-synthetic peptide antigen resin column to isolate antibodies that bind the phosphory A. WRN (Tyrosine 849). lated form of the site. After washing the column extensively, 0173 An 11 amino acid phospho-peptide antigen, the bound antibodies (i.e. antibodies that bind a phosphory DMESYy*QEIGR (where y-phosphotyrosine) that corre lated peptide described in A-C above, but do not bind the sponds to the sequence encompassing the tyrosine 849 phos non-phosphorylated form of the peptide) are eluted and kept phorylation site in human WRN chromatin or DNA binding/ in antibody storage buffer. repair/replication protein (see Row 51 of Table 1 (SEQ ID 0169. The isolated antibody is then tested for phospho NO: 50)), plus cysteine on the C-terminal for coupling, is specificity using Western blot assay using an appropriate cell constructed according to standard synthesis techniques using, line that expresses (or overexpresses) target phospho-protein e.g., a Rainin/Protein Technologies, Inc., Symphony peptide (i.e. phosphorylated VCP, HSP90B or TSN), for example, synthesizer. See ANTIBODIES: A LABORATORY MANUAL, CTV, CMK and MOLT15 cells, respectively. Cells are cul supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for tured in DMEM or RPMI supplemented with 10% FCS. Cell generation (and Subsequent Screening) of phospho-specific are collected, washed with PBS and directly lysed in cell lysis monoclonal WRN (tyr 849) antibodies as described in Immu buffer. The protein concentration of cell lysates is then mea nization/Fusion/Screening below. sured. The loading buffer is added into cell lysate and the mixture is boiled at 100° C. for 5 minutes. 20 ul (10 ug B. SPTA1 (Tyrosine 1538). protein) of sample is then added onto 7.5% SDS-PAGE gel. 0.174 An 11 amino acid phospho-peptide antigen, 0170 A standard Western blot may be performed accord DATNIQRKy*LK (where y-phosphotyrosine) that corre ing to the Immunoblotting Protocol set out in the CELL SIG sponds to the sequence encompassing the tyrosine 1538 phos NALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390. The phorylation site in human SPTA1 cytoskeletal protein (see isolated phospho-specific antibody is used at dilution 1:1000. Row 63 of Table 1 (SEQ ID NO: 62)), plus cysteine on the Phosphorylation-site specificity of the antibody will be C-terminal for coupling, is constructed according to standard shown by binding of only the phosphorylated form of the synthesis techniques using, e.g., a Rainin/Protein Technolo target protein. Isolated phospho-specific polyclonal antibody gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A does not (substantially) recognize the target protein when not LABORATORY MANUAL, supra.; Merrifield, supra. This pep phosphorylated at the appropriate phosphorylation site in the tide is then coupled to KLH and used to immunize animals non-stimulated cells (e.g. TSN is not bound when not phos and harvest spleen cells for generation (and Subsequent phorylated at tyrosine 210). screening) of phospho-specific monoclonal SPTA1 (tyr1538) 0171 In order to confirm the specificity of the isolated antibodies as described in Immunization/Fusion/Screening antibody, different cell lysates containing various phospho below. rylated signal transduction proteins other than the target pro C. SPTBN1 (tyrosine 1667). tein are prepared. The Western blot assay is performed again 0.175. A 15 amino acid phospho-peptide antigen, using these cell lysates. The phospho-specific polyclonal VDKLy*AGLKDLAEER (where y-phosphotyrosine) that antibody isolated as described above is used (1:1000 dilution) corresponds to the sequence encompassing the tyrosine 1667 to test reactivity with the different phosphorylated non-target phosphorylation site in human SPTBN1 cytoskeletal protein (see Row 61 of Table 1 (SEQID NO: 60), plus cysteine on the proteins on Western blot membrane. The phospho-specific C-terminal for coupling, is constructed according to standard antibody does not significantly cross-react with other phos synthesis techniques using, e.g., a Rainin/Protein Technolo phorylated signal transduction proteins, although occasion gies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A ally slight binding with a highly homologous phosphoryla LABORATORY MANUAL, supra.; Merrifield, supra. This pep tion-site on another protein may be observed. In Such case the tide is then coupled to KLH and used to immunize animals antibody may be further purified using affinity chromatogra and harvest spleen cells for generation (and Subsequent phy, or the specific immunoreactivity cloned by rabbit hybri screening) of phospho-specific monoclonal SPTBN1 doma technology. (tyr1667) antibodies as described in Immunization/Fusion/ Screening below. Example 3 Immunization/Fusion/Screening. Production of Phospho-Specific Monoclonal Anti 0176 A synthetic phospho-peptide antigenas described in bodies for the Detection of Target Signal Protein/ A-C above is coupled to KLH, and BALB/C mice are injected Polypepetide Phosphorylation intradermally (ID) on the back with antigen in complete Fre unds adjuvant (e.g. 50 ug antigen per mouse). The mice are 0172 Monoclonal antibodies that specifically bind a tar boosted with same antigen in incomplete Freund adjuvant get signal protein/polypepetide only when phosphorylated at (e.g. 25 ug antigen per mouse) every three weeks. After the the respective phosphorylation site disclosed herein (see fifth boost, the animals are sacrificed and spleens are har Table 1/FIG. 2) are produced according to standard methods vested. by first constructing a synthetic peptide antigen comprising (0177 Harvested spleen cells are fused to SP2/0 mouse the phosphorylation site sequence and then immunizing an myeloma fusion partner cells according to the standard pro animal to raise antibodies against the antigen, and harvesting tocol of Kohler and Milstein (1975). Colonies originating spleen cells from Such animals to produce fusion hybridomas, from the fusion are screened by ELISA for reactivity to the US 2010/0159477 A1 Jun. 24, 2010 36 phospho-peptide and non-phospho-peptide forms of the anti repair/replication protein (see Row 42 in Table 1 (SEQ ID gen and by Western blot analysis (as described in Example 1 NO: 41)), is constructed according to standard synthesis tech above). Colonies found to be positive by ELISA to the phos niques using, e.g., a Rainin/Protein Technologies, Inc., Sym pho-peptide while negative to the non-phospho-peptide are phony peptide synthesizer (see Merrifield, supra.) as further further characterized by Western blot analysis. Colonies described below in Synthesis & MS/MS Signature. The found to be positive by Western blot analysis are subcloned by TOP2B (tyr230) AQUA peptide is then spiked into a biologi limited dilution. Mouse ascites are produced from a single cal sample to quantify the amount of phosphorylated TOP2B clone obtained from Subcloning, and tested for phospho (tyr230) in the sample, as further described below in Analysis specificity (against the WRN, SFTA1 or SPTBN1 phospho & Quantification. peptide antigen, as the case may be) on ELISA. Clones iden tified as positive on Western blot analysis using cell culture C. PKCD (Tyrosine 374) Supernatant as having phospho-specificity, as indicated by a 0182 An AQUA peptide comprising the sequence strong band in the induced lane and a weak band in the GRGEy*FAIK (y-phosphotyrosine; sequence incorporat uninduced lane of the blot, are isolated and subcloned as clones producing monoclonal antibodies with the desired ing ''C/'N-labeled phenylalanine (indicated by bold F), specificity. which corresponds to the tyrosine374 phosphorylation site in human PKCD protein kinase (Ser/Thr) (see Row 123 in Table 0.178 Ascites fluid from isolated clones may be further 1 (SEQID NO: 122)), is constructed according to standard tested by Western blot analysis. The ascites fluid should pro synthesis techniques using, e.g., a Rainin/Protein Technolo duce similar results on Western blot analysis as observed gies, Inc., Symphony peptide synthesizer (see Merrifield, previously with the cell culture Supernatant, indicating phos supra.) as further described below in Synthesis & MS/MS pho-specificity against the phosphorylated target (e.g. SPTA1 Signature. The PKCD (tyr374) AQUA peptide is then spiked phosphorylated at tyrosine 1538). into a biological sample to quantify the amount of phospho rylated PKCD (tyr374) in the sample, as further described Example 4 below in Analysis & Quantification. Production and Use of Aqua Peptides for the Quanti fication of Target Signal Protein/Polypepetide Phos D. TAGLN3 (Tyrosine 192). phorylation 0183 An AQUA peptide comprising the sequence, (0179 Heavy-isotope labeled peptides (AQUA peptides GASQAGMTGy*GMPR (y-phosphotyrosine; sequence (internal standards)) for the detection and quantification of a incorporating ''C/'N-labeled proline (indicated by bold P), target signal protein/polypepetide only when phosphorylated which corresponds to the tyrosine 133 phosphorylation site in at the respective phosphorylation site disclosed herein (see human TAGLN3 cytoskeletal protein (see Row 66 in Table 1 Table 1/FIG. 2) are produced according to the standard (SEQID NO: 65)), is constructed according to standard syn AQUA methodology (see Gygi et al., Gerber et al., Supra.) thesis techniques using, e.g., a Rainin/Protein Technologies, methods by first constructing a synthetic peptide standard Inc., Symphony peptide synthesizer (see Merrifield, Supra.) corresponding to the phosphorylation site sequence and as further described below in Synthesis & MS/MS Signature. incorporating a heavy-isotope label. Subsequently, the MS' The TAGLN3 (tyr192) AQUA peptide is then spiked into a and LC-SRM signature of the peptide standard is validated, biological sample to quantify the amount of phosphorylated and the AQUA peptide is used to quantify native peptide in a TAGLN3 (tyr192) in the sample, as further described below biological sample, such as a digested cell extract. Production in Analysis & Quantification. and use of exemplary AQUA peptides is provided below. Synthesis & MS/MS Spectra. A. VASP (Tyrosine 15). 0.184 Fluorenylmethoxycarbonyl (Fmoc)-derivatized 0180. An AQUA peptide comprising the sequence, amino acid monomers may be obtained from AnaSpec (San ATVMLy*DDGNKR (y-phosphotyrosine; sequence incor Jose, Calif.). Fmoc-derivatized stable-isotope monomers porating 'C/N-labeled leucine (indicated by bold L). containing one 'N and five to nine C atoms may be which corresponds to the tyrosine 16 phosphorylation site in obtained from Cambridge Isotope Laboratories (Andover, human VASP cytoskeletal protein (see Row 79 in Table 1 Mass.). Preloaded Wang resins may be obtained from Applied (SEQID NO: 78)), is constructed according to standard syn Biosystems. Synthesis scales may vary from 5 to 25 umol. thesis techniques using, e.g., a Rainin/Protein Technologies, Amino acids are activated in situ with 1-H-benzotriazolium, Inc., Symphony peptide synthesizer (see Merrifield, Supra.) 1-bis(dimethylamino) methylene-hexafluorophosphate (1-), as further described below in Synthesis & MS/MS Signature. 3-oxide: 1-hydroxybenzotriazole hydrate and coupled at a The VASP (tyr 16) AQUA peptide is then spiked into a bio 5-fold molar excess over peptide. Each coupling cycle is logical sample to quantify the amount of phosphorylated followed by capping with acetic anhydride to avoid accumu lation of one-residue deletion peptide by-products. After syn VASP (tyr 16) in the sample, as further described below in thesis peptide-resins are treated with a standard Scavenger Analysis & Quantification. containing trifluoroacetic acid (TFA)-water cleavage B.TOP2B (Tyrosine 230). Solution, and the peptides are precipitated by addition to cold ether. Peptides (i.e. a desired AQUA peptide described in A-D 0181 An AQUA peptide comprising the sequence above) are purified by reversed-phase C18HPLC using stan IKHFDGEDy*TCITFTQPDLSK (y-phosphotyrosine; dard TFA/acetonitrile gradients and characterized by matrix sequence incorporating ''C/N-labeled leucine (indicated assisted laser desorption ionization-time of flight (Biflex III, by bold L), which corresponds to the tyrosine 230 phospho Bruker Daltonics, Billerica, Mass.) and ion-trap (Ther rylation site in human TOP2B chromatin or DNA binding/ moFinnigan, LCQ DecaXP) MS. US 2010/0159477 A1 Jun. 24, 2010 37
0185. MS/MS spectra for each AQUA peptide should applied to the complex mixture of peptides derived from exhibit a strong Y-type ion peak as the most intense fragment proteolytic cleavage of crude cell extracts to which the AQUA ion that is suitable for use in an SRM monitoring/analysis. peptides have been spiked in. 0187 LC-SRM of the entire sample is then carried out. Reverse-phase microcapillary columns (0.1 A-150-220 mm) MS/MS may be performed by using a ThermoFinnigan (San are prepared according to standard methods. An Agilent 1100 Jose, Calif.) mass spectrometer (LTQ ion trap or TSQ Quan liquid chromatograph may be used to develop and deliver a tum triple quadrupole). On the LTQ, parentions are isolated solvent gradient 10.4% acetic acid/0.005% heptafluorobu at 1.6 m/z, width, the ion injection time being limited to 100 tyric acid (HFBA)/7% methanol and 0.4% acetic acid/0. ms per microScan, with one microscans per peptide, and with 0.05% HFBA/65% methanol/35% acetonitrile) to the micro an AGC setting of 1x10; on the Quantum, Q1 is kept at 0.4 capillary column by means of a flow splitter. Samples are then and Q3 at 0.8 m/z, with a scan time of 200 ms per peptide. On directly loaded onto the microcapillary column by using a both instruments, analyte and internal standard are analyzed FAMOS inert capillary autosampler (LC Packings, San Fran in alternation within a previously known reverse-phase reten cisco) after the flow split. Peptides are reconstituted in 6% tion window; well-resolved pairs of internal standard and acetic acid/0.01% TFA before injection. analyte are analyzed in separate retention segments to improve duty cycle. Data are processed by integrating the Analysis & Quantification. appropriate peaks in an extracted ion chromatogram (60.15 m/z from the fragment monitored) for the native and internal 0186 Target protein (e.g. a phosphorylated protein of A-D standard, followed by calculation of the ratio of peak areas above) in a biological sample is quantified using a validated multiplied by the absolute amount of internal standard (e.g., AQUA peptide (as described above). The IAP method is then 384 fmol).
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 383
<21 Oc SEO ID NO 1 <211 LENGTH: 31 <212> TYPE PRT <213> ORGANISM: Homo sapiens <22 Os FEATURE; <221 NAME/KEY: MOD RES <222> LOCATION: (22) ... (22) OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs SEQUENCE: 1 Ala Lieu. Ser Glu Glu Met Ala Asp Thir Lieu. Glu Glu Gly Ser Ala Ser 1. 5 1O 15 Pro Thir Ser Pro Asp Tyr Ser Lieu. Asp Ser Pro Gly Pro Glu Lys 2O 25 3 O
SEO ID NO 2 LENGTH: 16 TYPE PRT ORGANISM: Homo sapiens FEATURE; NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs SEQUENCE: 2 Thir Ala Thr Asp Glu Ala Tyr Lys Asp Pro Ser Asn Lieu. Glin Gly Lys 1. 5 1O 15
<21 Os SEQ ID NO 3 &211s LENGTH: 2O 212s. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs SEQUENCE: 3 US 2010/0159477 A1 Jun. 24, 2010 38
- Continued Llys His Glu Ala Lieu Met Ser Asp Lieu. Ser Ala Tyr Gly Ser Ser Ile 1. 5 1O 15 Glin Ala Lieu. Arg 2O
<210s, SEQ ID NO 4 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 4 Glin Glin Val Ala Pro Thr Asp Asp Glu Thr Gly Lys Glu Lieu Val Lieu. 1. 5 1O 15 Ala Lieu. Tyr Asp Tyr Glin Glu Lys Ser Pro Arg 2O 25
<210s, SEQ ID NO 5 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 5 Gln Gly Phe Val Pro Ala Ala Tyr Val Lys 1. 5 1O
<210s, SEQ ID NO 6 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 6 Lieu. Glin Thr Ala Ser Asp Glu Ser Tyr Lys Asp Pro Thr Asn. Ile Glin 1. 5 1O 15
Ser Lys
<210s, SEQ ID NO 7 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 7 Llys Met Glu Glu Asp Ile Tyr Thr Asn Lieu. Ser Lys 1. 5 1O
<210s, SEQ ID NO 8 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/0159477 A1 Jun. 24, 2010 39
- Continued
22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 8 Ala Lieu Pro Ser Met Asp Llys Tyr Ala Val Phe Llys 1. 5 1O
<210s, SEQ ID NO 9 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 9 Lieu. Tyr Pro Glu Lieu. Ser Glin Tyr Met Gly Lieu Ser Lieu. Asn Glu Glu 1. 5 1O 15 Glu Ile Arg
<210s, SEQ ID NO 10 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 10 Ala Glu Gly Pro Thir Ile Lys Asn Asp Ala Ser Glin Glin Thr Asn Tyr 1. 5 1O 15 Gly Val Ala Val Lieu. Asp Llys Glu Ile Ile Glin Lieu. Ser Asp Tyr Lieu. 2O 25 3O Lys
<210s, SEQ ID NO 11 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 11 Ser Lieu. Glu Asn Gln Leu Tyr Thr Cys Thr Gln Lys Tyr Ser Pro Trp 1. 5 1O 15 Gly Met Lys
<210s, SEQ ID NO 12 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 12 US 2010/0159477 A1 Jun. 24, 2010 40
- Continued Ser Lieu. Glu Asn Gln Leu Tyr Thr Cys Thr Gln Lys Tyr Ser Pro Trp 1. 5 1O 15 Gly Met Lys
<210s, SEQ ID NO 13 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 13 Ser Glin Tyr Glu Ala Lieu Lys Glu Asp Trp Arg 1. 5 1O
<210s, SEQ ID NO 14 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 14 Ala Arg Tyr Asp Phe Cys Ala Arg 1. 5
<210s, SEQ ID NO 15 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 15 Trp Lieu. Glu Glin Phe Glu Met Ala Lieu. Ser Asn. Ile Arg Pro Asp Tyr 1. 5 1O 15 Ala Asp Ser Asn. Phe His Asp Phe Lys 2O 25
<210s, SEQ ID NO 16 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 16 Ala Arg Tyr Asp Phe Cys Ala Arg 1. 5
<210s, SEQ ID NO 17 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) US 2010/0159477 A1 Jun. 24, 2010 41
- Continued <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 17 Arg Gly Asp Ser Glin Pro Tyr Glin Ala Lieu Lys 1. 5 1O
<210s, SEQ ID NO 18 &211s LENGTH: 34 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 18 Ala Asp Gly Ala Thir Ser Asp Asp Lieu. Asp Lieu. His Asp Asp Arg Lieu 1. 5 1O 15 Ser Tyr Lieu Ser Ala Pro Gly Ser Glu Tyr Ser Met Tyr Ser Thr Asp 2O 25 3O Ser Arg
<210s, SEQ ID NO 19 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 19 Thr Lieu. Tyr Arg Ile Pro Glu Pro Gln Llys Pro Gln Leu Lys Pro Pro 1. 5 1O 15 Glu Asp Ile Val Arg 2O
<210s, SEQ ID NO 2 O &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O Glu Lys Glu Pro Glu Glu Glu Lieu. Tyr Asp Lieu. Ser Llys Val Val Lieu. 1. 5 1O 15 Ala Gly Gly Val Ala Pro Ala Lieu. Phe Arg 2O 25
<210s, SEQ ID NO 21 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 21 Pro Ser Pro Ala Ile Ser Val Ser Val Ser Ala Pro Ala Phe Tyr Ala US 2010/0159477 A1 Jun. 24, 2010 42
- Continued
1. 5 1O 15 Pro Glin Llys Llys 2O
<210s, SEQ ID NO 22 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 22 Asn Lieu. Tyr Pro Ser Ser Ser Pro Tyr Thr Arg 1. 5 1O
<210s, SEQ ID NO 23 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 23 Ala Asn Lieu. Glin Glin Ala Ile Val Thr Pro Leu Lys Pro Val Asp ASn 1. 5 1O 15 Thr Tyr Tyr Lys 2O
<210s, SEQ ID NO 24 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 24 Tyr Lys Tyr Arg Asp Lieu. Thr Val Arg Glu Thr Val Asn Val Ile Thr 1. 5 1O 15 Lieu. Tyr Lys
<210s, SEQ ID NO 25 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 25 Tyr Lys Tyr Arg Asp Lieu. Thr Val Arg Glu Thr Val Asn Val Ile Thr 1. 5 1O 15 Lieu. Tyr Lys
<210s, SEQ ID NO 26 &211s LENGTH: 12 212. TYPE: PRT US 2010/0159477 A1 Jun. 24, 2010 43
- Continued <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 26 Gly Ile Leu Lleu Tyr Gly Pro Pro Gly Thr Gly Lys 1. 5 1O
<210s, SEQ ID NO 27 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 27 Lieu. Asp Gln Lieu. Ile Tyr Ile Pro Lieu Pro Asp Glu Lys 1. 5 1O
<210s, SEQ ID NO 28 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 28 Asn Cys Lieu Val Tyr Ser Ile Pro Thr Asn Ser Ser Lys 1. 5 1O
<210s, SEQ ID NO 29 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 29 Val Ile Lieu. His Lieu Lys Glu Asp Glin Thr Glu Tyr Lieu. Glu Glu Arg 1. 5 1O 15
<210s, SEQ ID NO 3 O &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 30 Glu Lieu. Asp Pro Thr Asn Met Thr Tyr Ile Thr Asn Glin Ala Ala Val 1. 5 1O 15 Tyr Phe Glu Lys Gly Asp Tyr Asn Lys 2O 25
<210s, SEQ ID NO 31 US 2010/0159477 A1 Jun. 24, 2010 44
- Continued
&211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (23) . . (23) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 31 Glu Lieu. Asp Pro Thr Asn Met Thr Tyr Ile Thr Asn Glin Ala Ala Val 1. 5 1O 15 Tyr Phe Glu Lys Gly Asp Tyr Asn Lys 2O 25
<210s, SEQ ID NO 32 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 32 Ala Ala Ala Lieu. Glu Ala Met Lys Asp Tyr Thr Lys 1. 5 1O
<210 SEQ ID NO 33 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 33 Met Arg Ala Glu Asp Gly Glu Asn Tyr Asp Ile Llys Llys 1. 5 1O
<210s, SEQ ID NO 34 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 34 Ile Lieu. Glu Asn. Glu Lys Asp Lieu. Glu Glu Ala Glu Glu Tyr Lys Glu 1. 5 1O 15 Ala Arg
<210s, SEQ ID NO 35 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 35 Lys Asp Tyr Asn. Glu Ala Tyr Asn Tyr Tyr Thr Lys US 2010/0159477 A1 Jun. 24, 2010 45
- Continued
<210s, SEQ ID NO 36 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 36 Llys Lieu. Asp Asp Ala Ile Glu Asp Cys Thr Asn Ala Wall Lys Lieu. Asp 1. 5 1O 15 Asp Thr Tyr Ile Lys 2O
<210s, SEQ ID NO 37 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 37 Arg Pro Trp Val Glu Tyr Glu ASn Val Arg Gln Glu Tyr Glu Glu Val 1. 5 1O 15 Lys
<210s, SEQ ID NO 38 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 38 Ser Met Met Ser Ala Tyr Glu Arg 1. 5
<210s, SEQ ID NO 39 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 39 Ser Met Met Ser Ser Tyr Ser Ala Ala Asp Arg 1. 5 1O
<210s, SEQ ID NO 4 O &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine US 2010/0159477 A1 Jun. 24, 2010 46
- Continued
<4 OOs, SEQUENCE: 4 O Glu Ala Asp Ser Val Tyr Gly Glu Trp Val Pro Val Glu Lys 1. 5 1O
<210s, SEQ ID NO 41 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 41 Ile Llys His Phe Asp Gly Glu Asp Tyr Thr Cys Ile Thr Phe Glin Pro 1. 5 1O 15 Asp Lieu. Ser Lys 2O
<210s, SEQ ID NO 42 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 42 His Val Asp Tyr Val Val Asp Glin Val Val Gly Lys 1. 5 1O
<210s, SEQ ID NO 43 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 43 Llys Val Glu Glu Val Val Tyr Asp Lieu. Ser Ile Arg 1. 5 1O
<210s, SEQ ID NO 44 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 44 Glin Val Tyr Asp Gly Phe Ser Phe Ile Gly Asn Thr Gly Pro Tyr Glu 1. 5 1O 15 Val Ser Lys Llys 2O
<210s, SEQ ID NO 45 &211s LENGTH: 14 212. TYPE: PRT US 2010/0159477 A1 Jun. 24, 2010 47
- Continued <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 45 Gly Gly Pro Lieu. Asn Lys Glu Tyr Thr Asn Trp Glu Ala Lys 1. 5 1O
<210s, SEQ ID NO 46 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 46 Asp Met Glu Ser Asp Tyr Ser Gly Glin Gly Val Asp Gln Lieu. Glin Arg 1. 5 1O 15
<210s, SEQ ID NO 47 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 47 Llys Phe Asn His Asp Gly Glu Glu Glu Glu Glu Asp Asp Asp Tyr Gly 1. 5 1O 15 Ser Arg
<210s, SEQ ID NO 48 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 48 Arg Arg Asp Glu Asp Met Lieu. Tyr Ser Pro Glu Lieu Ala Glin Arg 1. 5 1O 15
<210s, SEQ ID NO 49 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 49 Asp Asp Val Tyr Glu Ala Phe Met Lys 1. 5
<210s, SEQ ID NO 50 &211s LENGTH: 11 US 2010/0159477 A1 Jun. 24, 2010 48
- Continued
212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 50 Asp Met Glu Ser Tyr Tyr Glin Glu Ile Gly Arg 1. 5 1O
<210s, SEQ ID NO 51 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 51 Arg Val Val Ser Glu Asp Thir Ser His Tyr Ile Lieu. Ile Llys 1. 5 1O
<210s, SEQ ID NO 52 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 52 Llys Phe Thr Tyr Lieu. Gly Ser Glin Asp Arg 1. 5 1O
<210s, SEQ ID NO 53 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 53 Phe Lieu. Glu Asn Gly Ser Glin Glu Asp Lieu. Lieu. His Gly Asn Pro Gly 1. 5 1O 15 Ser Thr Tyr Lieu Ala Ser Asn Ser Thr Ser Ala Pro Asn Trp Llys 2O 25 3O
<210s, SEQ ID NO 54 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 54 Asp Ala Ile Tyr Ser His Lys Asn. Cys Pro Tyr Asp Ala Lys 1. 5 1O US 2010/0159477 A1 Jun. 24, 2010 49
- Continued <210s, SEQ ID NO 55 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 55 Glin Val Lieu Val Lys Ala Glu Lieu. Glu Lys Tyr Glin Glin Tyr Lys 1. 5 1O 15
<210s, SEQ ID NO 56 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 56 Asp Ala Thir Asn. Ile Glin Arg Llys Tyr Lieu Lys 1. 5 1O
<210s, SEQ ID NO 57 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 57 Lys Gly Tyr Val Ser Lieu. Glu Asp Tyr Thr Ala Phe Lieu. Ile Asp Llys 1. 5 1O 15 Glu Ser Glu Asn. Ile Llys 2O
<210s, SEQ ID NO 58 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 58 Ile Thr Asp Lieu. Tyr Thr Asp Lieu. Arg 1. 5
<210s, SEQ ID NO 59 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 59 Met Lieu. Thir Ala Glin Asp Met Ser Tyr Asp Glu Ala Arg 1. 5 1O US 2010/0159477 A1 Jun. 24, 2010 50
- Continued
<210s, SEQ ID NO 60 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 60 Val Asp Llys Lieu. Tyr Ala Gly Lieu Lys Asp Lieu Ala Glu Glu Arg 1. 5 1O 15
<210s, SEQ ID NO 61 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 61 Thir Ser Ser Ile Ser Gly Pro Leu Ser Pro Ala Tyr Thr Gly Glin Val 1. 5 1O 15 Pro Tyr Asn Tyr Asn Glin Lieu. Glu Gly Arg 2O 25
<210s, SEQ ID NO 62 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 62 Thir Ser Ser Ile Ser Gly Pro Leu Ser Pro Ala Tyr Thr Gly Glin Val 1. 5 1O 15 Pro Tyr Asn Tyr Asn Glin Lieu. Glu Gly Arg 2O 25
<210s, SEQ ID NO 63 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 63 Glu Val Val Ala Ala Ser His Glu Lieu. Gly Glin Asp Tyr Glu. His Val 1. 5 1O 15 Thr Met Leu Arg 2O
<210s, SEQ ID NO 64 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: US 2010/0159477 A1 Jun. 24, 2010 51
- Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 64 Asn Ser Leu Glin Asn Lieu. Ser Glin Tyr Pro Met Met Arg 1. 5 1O
<210s, SEQ ID NO 65 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 65 Gly Ala Ser Glin Ala Gly Met Thr Gly Tyr Gly Met Pro Arg 1. 5 1O
<210s, SEQ ID NO 66 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 66 Lys Gly Ile Trp Lieu. Glu Ala Gly Lys Ala Lieu. Asp Tyr Tyr Met Lieu. 1. 5 1O 15 Arg
<210s, SEQ ID NO 67 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 67 Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg 1. 5 1O 15
<210s, SEQ ID NO 68 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 68 Val Glin Ile Val Tyr Llys Pro Val Asp Lieu. Ser Lys 1. 5 1O
<210s, SEQ ID NO 69 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/0159477 A1 Jun. 24, 2010 52
- Continued
22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 69 Lieu. Gly Val Ile Val Ser Ala Tyr Met His Tyr Ser Lys 1. 5 1O
<210s, SEQ ID NO 70 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 7 O Lieu. Gly Val Ile Val Ser Ala Tyr Met His Tyr Ser Lys 1. 5 1O
<210s, SEQ ID NO 71 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 71 Arg Thr Glin Tyr Ser Cys Tyr Cys Cys Llys 1. 5 1O
<210s, SEQ ID NO 72 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 72 Asp Arg Glu Asp Tyr Val Pro Tyr Thr Gly Glu Lys 1. 5 1O
<210s, SEQ ID NO 73 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 73 His Ile Ala Glu Glu Ala Asp Arg Llys Tyr Glu Glu Val Ala Arg 1. 5 1O 15
<210s, SEQ ID NO 74 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: US 2010/0159477 A1 Jun. 24, 2010 53
- Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 74 Ile Asin Val Tyr Tyr Asn. Glu Ala Thr Gly Gly Lys 1. 5 1O
<210s, SEQ ID NO 75 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 75 Llys Lieu. Lieu. Ile Ala Glin Glu Pro Lieu. Tyr Glin Cys 1. 5 1O
<210s, SEQ ID NO 76 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 76 Lieu. His Tyr Pro Met Val Glu Tyr Cys Ile Pro Thr Thr Ser Gly Glu 1. 5 1O 15 Asp Val Arg Asp Phe Thr Lys 2O
<210s, SEQ ID NO 77 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 77 Lieu. His Tyr Pro Met Val Glu Tyr Cys Ile Pro Thr Thr Ser Gly Glu 1. 5 1O 15 Asp Val Arg
<210s, SEQ ID NO 78 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 78 Ala Thr Val Met Lieu. Tyr Asp Asp Gly Asn Lys Arg 1. 5 1O
<210s, SEQ ID NO 79 US 2010/0159477 A1 Jun. 24, 2010 54
- Continued
&211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 79 Ser Ser Ser Ser Val Thir Thr Ser Glu Thr Gln Pro Cys Thr Pro Ser 1. 5 1O 15 Ser Ser Asp Tyr Ser Asp Lieu. Glin Arg 2O 25
<210s, SEQ ID NO 8O &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 80 Asn Lieu. Glin Glu Ala Glu Glu Trp Tyr Lys 1. 5 1O
<210 SEQ ID NO 81 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 81 Phe Tyr Thr Asn Pro Ser Tyr Phe Phe Asp Leu Trp Llys 1. 5 1O
<210s, SEQ ID NO 82 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 82 Tyr Glu Tyr Glin Pro Phe Ala Gly Lys 1. 5
<210s, SEQ ID NO 83 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 83 Met Tyr Val Pro Met Thr Glu Asp Ile Tyr Asn Ala Ile Ser Ala Lys 1. 5 1O 15 US 2010/0159477 A1 Jun. 24, 2010 55
- Continued
<210s, SEQ ID NO 84 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 84 Met Tyr Val Pro Met Thr Glu Asp Ile Tyr Asn Ala Ile Ser Ala Lys 1. 5 1O 15
<210s, SEQ ID NO 85 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 85 Lieu. Gly Glin Ser Asp Pro Ala Pro Lieu Gln His Glin Met Asp Ile Tyr 1. 5 1O 15 Glin Lys
<210s, SEQ ID NO 86 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 86 His Val Asp Tyr Val Ala Asp Glin Ile Val Thir Lys 1. 5 1O
<210s, SEQ ID NO 87 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 87 Tyr Asn Pro Tyr Thr Arg Ser Ile Glin Ile Leu Lys 1. 5 1O
<210s, SEQ ID NO 88 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 88 Tyr Asn Pro Tyr Thr Arg Ser Ile Glin Ile Leu Lys 1. 5 1O US 2010/0159477 A1 Jun. 24, 2010 56
- Continued
<210s, SEQ ID NO 89 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 89 Val Pro Ala Asp Thr Glu Val Val Cys Ala Pro Pro Thr Ala Tyr Ile 1. 5 1O 15 Asp Phe Ala Arg 2O
<210s, SEQ ID NO 90 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 90 Val Val Lieu Ala Tyr Glu Pro Val Trp Ala Ile Gly Thr Gly Lys 1. 5 1O 15
<210s, SEQ ID NO 91 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 91 Gly Met Tyr Asp Val Gly Lieu Pro Ser Arg 1. 5 1O
<210s, SEQ ID NO 92 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 92 Thr Asn Pro Thr Glu Pro Val Gly Val Val Cys Arg Val Asp Gly Val 1. 5 1O 15 Tyr Glin Val Val Glu Tyr Ser Glu Ile Ser Leu Ala Thr Ala Gln Lys 2O 25 3O
<210s, SEQ ID NO 93 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine US 2010/0159477 A1 Jun. 24, 2010 57
- Continued
<4 OOs, SEQUENCE: 93 Gly Gly. Thir Lieu. Thr Glin Tyr Glu Gly Lys Lieu. Arg 1. 5 1O
<210s, SEQ ID NO 94 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 94 Ala Ala Lieu. Gly Pro Lieu Val Thr Gly Lieu. Tyr Asp Val Glin Ala Phe 1. 5 1O 15 Lys
<210s, SEQ ID NO 95 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 95 Tyr Ile Ala Asn Lieu. Gly His Gly Lieu. Tyr Pro Asp Met Asp Pro Glu 1. 5 1O 15 His Val Gly Ala Phe Val Asp Ala Wal His Lys 2O 25
<210s, SEQ ID NO 96 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 96 Ala Ala Trp Gly Glu Glu Thir Asp Tyr Thr Pro Val Trp Cys Met Arg 1. 5 1O 15
<210s, SEQ ID NO 97 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 97 Ser Lieu. Gly Asn Val Ile Asp Pro Lieu. Asp Val Ile Tyr Gly Ile Ser 1. 5 1O 15 Lieu. Glin Gly Lieu. His Asn Gln Lieu. Lieu. Asn. Ser Asn Lieu. Asp Pro Ser 2O 25 3O Glu Val Glu Lys Ala Lys 35 US 2010/0159477 A1 Jun. 24, 2010 58
- Continued
<210s, SEQ ID NO 98 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 98 Ser His Ile Lieu. Tyr Asp Arg Ala Gly Glu Glu. His Tyr Asn. Cys Ile 1. 5 1O 15 Ser Ala Lieu. His Llys 2O
<210s, SEQ ID NO 99 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 99 Glu Phe Ile Glu Ser Lieu Gln Lieu Lys Pro Gly Glin Val Val Tyr Lys 1. 5 1O 15
<210s, SEQ ID NO 100 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 1.OO Ala Val Phe Gly His Pro Phe Ser Leu Gly Trp Ala Ser Pro Phe Ala 1. 5 1O 15 Thr Pro Asp Glin Gly Lys Ala Asp Pro Tyr Glin Tyr Val Val 2O 25 3O
<210s, SEQ ID NO 101 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 101 Val Lieu. Asn. Glu Lieu. Tyr Ser Val Met Lys 1. 5 1O
<210s, SEQ ID NO 102 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine US 2010/0159477 A1 Jun. 24, 2010 59
- Continued
<4 OOs, SEQUENCE: 102 Tyr Pro Lieu. Ile Ile Val Ser Glu Glu Ile Glu Tyr Lieu. Lieu Lys 1. 5 1O 15
<210s, SEQ ID NO 103 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 103 Gly Asn Lieu. Tyr Ser Phe Gly Cys Pro Glu Tyr Gly Gln Leu Gly His 1. 5 1O 15 Asn Ser Asp Gly Lys 2O
<210s, SEQ ID NO 104 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 104 Val Phe Ser Trp Gly Phe Gly Gly Tyr Gly Arg 1. 5 1O
<210s, SEQ ID NO 105 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 105 Val Gly Trp Phe Pro Ala Asn Tyr Val Glu Glu Asp Tyr Ser Glu Tyr 1. 5 1O 15 Cys
<210s, SEQ ID NO 106 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 106 Lieu. Tyr Pro Val Ser Arg Phe 1. 5
<210s, SEQ ID NO 107 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 2010/0159477 A1 Jun. 24, 2010 60
- Continued
22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 107 Met Val Val Lieu. Glu. Cys Met Lieu Lys Lys Asp Lieu. Ile Tyr Asn Lys 1. 5 1O 15
<210s, SEQ ID NO 108 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 108 Asp Asn. Cys Glin Tyr Val Tyr Asn Val Asp Glin Arg 1. 5 1O
<210s, SEQ ID NO 109 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 109 Asp Glin Asp Glu Lieu. Asn Pro Tyr Ala Ala Trp Arg 1. 5 1O
<210s, SEQ ID NO 110 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 110 Tyr Phe Val Asp Lys Ala Ala Ala Arg Llys Val Lieu. Ser Arg 1. 5 1O
<210s, SEQ ID NO 111 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 111 Ala Glin Asp Glu Ile Pro Ala Lieu. Ser Val Ser Arg Pro Glin Thr Gly 1. 5 1O 15 Lieu. Ser Phe Lieu. Gly Pro Glu Pro Glu Asp Lieu. Glu Asp Lieu. Tyr Ser 2O 25 3O Arg US 2010/0159477 A1 Jun. 24, 2010 61
- Continued <210s, SEQ ID NO 112 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 112 Gly Val Lieu Met Tyr Gly Pro Pro Gly Cys Gly Lys 1. 5 1O
<210s, SEQ ID NO 113 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 113 Lys Asp Glu Glin Glu. His Glu Phe Tyr Lys 1. 5 1O
<210s, SEQ ID NO 114 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 114 Val Asn Glu Ser Ser His Tyr Asp Leu Ala Phe Thr Asp Val His Phe 1. 5 1O 15 Llys Pro Gly Glin Ile Arg 2O
<210s, SEQ ID NO 115 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 115 Lieu. Asp Ser Ser Asp Ile Tyr Asn. Glu Lieu Lys Glu Thir Tyr Pro Asn 1. 5 1O 15 Tyr Lieu Pro Lieu. Tyr Val Ala Arg 2O
<210s, SEQ ID NO 116 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 116 US 2010/0159477 A1 Jun. 24, 2010 62
- Continued
Gly Glu Val Lieu. Glu Gly Ser Asn Ala Tyr Tyr Cys Glu Lys 1. 5 1O
<210s, SEQ ID NO 117 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 117 Val Tyr Asp Gln Thr Asn Pro Tyr Thr Asp Val Arg 1. 5 1O
<210s, SEQ ID NO 118 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 118 Cys Cys Met Pro Tyr Thr Pro Ile Cys Ile Ala Lys 1. 5 1O
<210s, SEQ ID NO 119 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 119 Val Phe Pro Gly Ser Thr Thr Glu Asp Tyr Asn Lieu. Ile Val Ile Glu 1. 5 1O 15 Arg
<210s, SEQ ID NO 120 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 120 Thr Lieu. Tyr Glu. His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser 1. 5 1O 15
Ser Lys
<210s, SEQ ID NO 121 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) US 2010/0159477 A1 Jun. 24, 2010 63
- Continued <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 121 Thr Lieu. Tyr Glu. His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser 1. 5 1O 15 Ser Lys
<210s, SEQ ID NO 122 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 122 Gly Arg Gly Glu Tyr Phe Ala Ile Llys 1. 5
<210s, SEQ ID NO 123 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 123 Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg 1. 5 1O
<210s, SEQ ID NO 124 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 124 Llys Met Ser Tyr Ser Gly Lys Glin Ser Asn. Glu Lys 1. 5 1O
<210s, SEQ ID NO 125 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 125 Pro Ile Glu Asp Val Thir Ile Tyr Glu Lys 1. 5 1O
<210s, SEQ ID NO 126 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0159477 A1 Jun. 24, 2010 64
- Continued <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 126 Ile Thr Gly Tyr Ile Val Glu Lys 1. 5
<210s, SEQ ID NO 127 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 127 Thir Lieu. Glu Lieu. Cys Val Asp Asn Lieu. Glin Pro Asp Phe Lieu. Tyr Asp 1. 5 1O 15 His Ile Glin Pro Val Arg 2O
<210s, SEQ ID NO 128 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 128 Gly His Asn Gly Lys Ile Tyr Pro Tyr Lieu Val Met Asn Asp Ala Cys 1. 5 1O 15 Lieu. Thr Glu Ser Arg 2O
<210s, SEQ ID NO 129 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 129 Asp Asp Tyr Gly Phe Ser Gly Ser Glin Lys 1. 5 1O
<210s, SEQ ID NO 130 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 130 Ile Gly Lys Gly Ser Phe Gly Glu Val Tyr Lys 1. 5 1O
<210s, SEQ ID NO 131 US 2010/0159477 A1 Jun. 24, 2010 65
- Continued
&211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 131 Asn Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ala Asp Ser Ile Glin 1. 5 1O 15 Ala Glu Glu Trp Tyr Phe Gly Lys 2O
<210s, SEQ ID NO 132 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 132 Llys Lieu. Tyr Ser Gly Ala Glin Thr Asp Gly Lys 1. 5 1O
<210 SEQ ID NO 133 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 133 Lys Glu Glin Gly Thr Tyr Ala Lieu. Ser Lieu. Ile Tyr Gly Lys 1. 5 1O
<210s, SEQ ID NO 134 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 134 Met Arg Ala Cys Tyr Tyr Ser Lieu Ala Ser Lys 1. 5 1O
<210s, SEQ ID NO 135 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 135 Met Arg Ala Cys Tyr Tyr Ser Lieu Ala Ser Lys 1. 5 1O US 2010/0159477 A1 Jun. 24, 2010 66
- Continued
<210s, SEQ ID NO 136 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 136 Asn. Cys Asp Asp Glu Val Tyr Glu Lieu Met Arg 1. 5 1O
<210s, SEQ ID NO 137 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 137 Val Phe Lieu Ala Glu. Cys Tyr Asn Lieu. Ser Pro Thir Lys Asp Llys 1. 5 1O 15
<210s, SEQ ID NO 138 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 138 Glu Val Tyr Asp Wal Met Lieu. Gly Cys Trp Glin Arg 1. 5 1O
<210s, SEQ ID NO 139 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 139 Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Glin Gly Cys Ala Ser Lys Lieu. 1. 5 1O 15 Pro Val Lys
<210s, SEQ ID NO 140 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 140 Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Glin Gly Cys Ala Ser Lys Lieu. 1. 5 1O 15 US 2010/0159477 A1 Jun. 24, 2010 67
- Continued
Pro Val Lys
<210s, SEQ ID NO 141 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 141 Lieu Val Gly Asp Thr Val Ser Tyr Ser Lys 1. 5 1O
<210s, SEQ ID NO 142 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 142 Asn Ile Phe Val Ala Trp Phe Val Thr Val Pro Val Ala Gly Lieu. Phe 1. 5 1O 15 Ser Ala Ala Val Met Ala Lieu. Leu Met Tyr Gly Ile Leu Pro Tyr Val 2O 25 3O
<210s, SEQ ID NO 143 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (31) ... (31) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 143 Asn Ile Phe Val Ala Trp Phe Val Thr Val Pro Val Ala Gly Lieu. Phe 1. 5 1O 15 Ser Ala Ala Val Met Ala Lieu. Leu Met Tyr Gly Ile Leu Pro Tyr Val 2O 25 3O
<210s, SEQ ID NO 144 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 144 Gly Ser Gly Ser Val Val Gly Glu Lieu Met Tyr Lys 1. 5 1O
<210s, SEQ ID NO 145 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0159477 A1 Jun. 24, 2010 68
- Continued <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 145 Phe Ser Ile Ser Pro Asp Glu Asp Ser Ser Ser Tyr Ser Ser Asn Ser 1. 5 1O 15 Asp Phe Asn Tyr Ser Tyr Pro Thr Lys 2O 25
<210s, SEQ ID NO 146 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 146 Ala Val Tyr Ile Gly Val His Val Pro Phe Ser Lys 1. 5 1O
<210s, SEQ ID NO 147 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 147 Ile Gly Ser Ser Lieu. Tyr Ala Lieu. Gly Thr Glin Asp Ser Thr Asp Ile 1. 5 1O 15 Cys Llys
<210s, SEQ ID NO 148 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (23) . . (23) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 148 Ser Asn Val Ile Val Ala Lieu Ala Arg Asp Ser Lieu Ala Lieu Ala Arg 1. 5 1O 15 Pro Llys Ser Ser Asp Val Tyr Val Ser Tyr Asp Tyr Gly Lys Ser Phe 2O 25 3O Lys
<210s, SEQ ID NO 149 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (26) ... (26) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 149 Ser Asn Val Ile Val Ala Lieu Ala Arg Asp Ser Lieu Ala Lieu Ala Arg US 2010/0159477 A1 Jun. 24, 2010 69
- Continued
1. 5 1O 15 Pro Llys Ser Ser Asp Val Tyr Val Ser Tyr Asp Tyr Gly Lys Ser Phe 2O 25 3O Lys
<210s, SEQ ID NO 150 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 150 Arg Glu Val Ala Pro Pro Tyr Glin Gly Ala Asp Pro Ile Lieu Ala Thr 1. 5 1O 15 Ala Lieu Ala Ser Asp Pro Ile Pro Asn Pro Lieu Gln Lys 2O 25
<210s, SEQ ID NO 151 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 151 Trp Glu Asp Ser Ala His Llys Pro Glin Ser Lieu. Asp Thr Asp Asp Pro 1. 5 1O 15 Ala Thr Lieu. Tyr Ala Val Val Glu Asn Val Pro Pro Lieu. Arg 2O 25 3O
<210s, SEQ ID NO 152 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 152 Ile Asin Glu Ser Lieu. Arg Asp Gln Lieu. Lieu Val Thir Ile Glin Llys Thr 1. 5 1O 15 Phe Thr Tyr
<210s, SEQ ID NO 153 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 153 Ala Ser Lieu. Arg Asp Asn Lieu. Tyr Lieu. Lieu. Glin Ala Phe Met Tyr Ile 1. 5 1O 15 Lieu. Gly Ile Cys Lieu US 2010/0159477 A1 Jun. 24, 2010 70
- Continued
<210s, SEQ ID NO 154 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 154 Pro Pro Tyr Tyr Pro Gln Pro Glu Asn Pro Asp Ser Gly Gly Asn Ile 1. 5 1O 15 Tyr Pro Arg Pro Llys Pro Arg 2O
<210s, SEQ ID NO 155 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 155 Lys Arg Tyr Asp Gln Asp Lieu. Cys Tyr Thr Asp Ile Lieu. Phe Thr Glu. 1. 5 1O 15 Glin Glu Arg
<210s, SEQ ID NO 156 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 156 Met Lieu Gln His Ile Asp Tyr Arg 1. 5
<210s, SEQ ID NO 157 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 157 Ala Ala Phe Gly Ile Ser Asp Ser Tyr Val Asp Gly Ser Ser Phe Asp 1. 5 1O 15 Pro Glin Arg
<210s, SEQ ID NO 158 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0159477 A1 Jun. 24, 2010 71
- Continued <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 158 Phe Glin Ser Asp Ser Ser Ser Tyr Pro Thr Val Asp Ser Asn Ser Lieu. 1. 5 1O 15 Lieu. Gly Glin Ser Arg 2O
<210s, SEQ ID NO 159 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 159 Val Pro Lieu. Ser Ala Tyr Glu Arg 1. 5
<210s, SEQ ID NO 160 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 160 Ser Pro Tyr Ser Arg Ser Arg Tyr Arg Glu Ser Arg Tyr Gly Gly Ser 1. 5 1O 15 His Tyr Ser Ser Ser Gly Tyr Ser Asn Ser Arg 2O 25
<210s, SEQ ID NO 161 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 161 Ser Pro Tyr Ser Arg Ser Arg Tyr Arg Glu Ser Arg Tyr Gly Gly Ser 1. 5 1O 15 His Tyr Ser Ser Ser Gly Tyr Ser Asn Ser Arg 2O 25
<210s, SEQ ID NO 162 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 162 Ser Pro Tyr Ser Arg Ser Arg Tyr Arg Glu Ser Arg Tyr Gly Gly Ser 1. 5 1O 15 US 2010/0159477 A1 Jun. 24, 2010 72
- Continued
His Tyr Ser Ser Ser Gly Tyr Ser Asn Ser Arg 2O 25
<210s, SEQ ID NO 163 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 163 Gly Ile Lieu. His Leu Ser Pro Asp Val Tyr Glin Glu Met Glu Ala Ser 1. 5 1O 15 Arg
<210s, SEQ ID NO 164 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 164 Ser Glin Ser Tyr Gly Gly Tyr Glu Asn Glin Lys Glin Ser Ser Tyr 1. 5 1O 15
<210s, SEQ ID NO 165 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 165 Gly Gly Tyr Gly Gly Asp Arg Gly Gly Gly Tyr Gly Gly Asp Arg Gly 1. 5 1O 15 Gly Tyr Gly Gly Asp Arg Gly Gly Tyr Gly Gly Asp Arg 2O 25
<210s, SEQ ID NO 166 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 166 Phe Thr Glu Tyr Glu Thr Glin Val Lys 1. 5
<210s, SEQ ID NO 167 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES US 2010/0159477 A1 Jun. 24, 2010 73
- Continued <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 167 Ile His Ala Tyr Val Asp Tyr Pro Ile Tyr Asp Val Lieu Gln Met Val 1. 5 1O 15 Gly His Ala Asn Arg Pro Lieu. Glin Asp Asp Glu Gly Arg 2O 25
<210s, SEQ ID NO 168 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 168 Arg Met Thr Glin Asn Pro Asn Tyr Tyr Asn Lieu Gln Gly Ile Ser His 1. 5 1O 15 Arg
<210s, SEQ ID NO 169 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 169 Lys Ser Lieu. Glu Val Trp Thr Lys Asp His Pro Phe Pro Ile Asp Ile 1. 5 1O 15 Asp Tyr Met Ile Ser Asp Thir Lieu. Glu Lieu. Lieu. Arg 2O 25
<210s, SEQ ID NO 170 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 170 Val Gly. Thir Ile Asp Asp Asp Pro Glu Tyr Arg 1. 5 1O
<210s, SEQ ID NO 171 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 171 Asn Lys Asp Arg Pro Ala Met Glin Lieu. Tyr Glin Pro Gly Ala Arg 1. 5 1O 15 US 2010/0159477 A1 Jun. 24, 2010 74
- Continued
<210s, SEQ ID NO 172 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 172 Thr Gly Tyr Gly Gly Gly Phe Asin Glu Arg 1. 5 1O
<210s, SEQ ID NO 173 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 173 Glu Asn Val Glu Tyr Ile Glu Arg Glu Glu Ser Asp Gly Glu Tyr Asp 1. 5 1O 15 Glu Phe Gly Arg Llys 2O
<210s, SEQ ID NO 174 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 174 Glu Asn Val Glu Tyr Ile Glu Arg Glu Glu Ser Asp Gly Glu Tyr Asp 1. 5 1O 15 Glu Phe Gly Arg Llys 2O
<210s, SEQ ID NO 175 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 175 Ala Tyr His Asn. Ser Pro Ala Tyr Lieu Ala Tyr Ile Asn Ala Lys 1. 5 1O 15
<210s, SEQ ID NO 176 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine US 2010/0159477 A1 Jun. 24, 2010 75
- Continued <4 OOs, SEQUENCE: 176 Ser Ala Val Tyr Pro Lieu. Lieu. Tyr Arg 1. 5
<210s, SEQ ID NO 177 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 177 Thr Pro Glin Gly Arg Glu Tyr Gly Met Ile Tyr Lieu. Gly Lys Asp Thr 1. 5 1O 15 Asn Gly Glu Asn. Ile Ala Glu Ser Lieu Val Ala Glu Gly Lieu Ala Thr 2O 25 3O Arg
<210s, SEQ ID NO 178 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) . . (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 178 Asp Glin Arg Glu Glu Glu Lieu. Gly Glu Tyr Tyr Met Lys 1. 5 1O
<210s, SEQ ID NO 179 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 179 Gly Ile Tyr Lys Asp Asp Ile Ala Glin Val Asp Tyr Val Glu Pro Ser 1. 5 1O 15 Glin Asn. Thir Ile Ser Lieu Lys 2O
<210s, SEQ ID NO 18O &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 18O Thr Pro Met Tyr Gly Ser Glin Thr Pro Met Tyr Gly Ser Gly Ser Arg 1. 5 1O 15
<210s, SEQ ID NO 181 &211s LENGTH: 15 US 2010/0159477 A1 Jun. 24, 2010 76
- Continued
212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 181 Thr Pro Met Tyr Gly Ser Glin Thr Pro Leu Gln Asp Gly Ser Arg 1. 5 1O 15
<210s, SEQ ID NO 182 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 182 Tyr Pro Asp Gly His Gly Asp Tyr Ala Tyr Glin Glin Ser Ser Tyr Thr 1. 5 1O 15 Glu Glin Ser Tyr Asp Arg Ser Phe 2O
<210s, SEQ ID NO 183 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 183 Tyr Pro Asp Gly His Gly Asp Tyr Ala Tyr Glin Glin Ser Ser Tyr Thr 1. 5 1O 15 Glu Glin Ser Tyr Asp Arg Ser Phe 2O
<210s, SEQ ID NO 184 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 184 Tyr Pro Asp Gly His Gly Asp Tyr Ala Tyr Glin Glin Ser Ser Tyr Thr 1. 5 1O 15 Glu Glin Ser Tyr Asp Arg Ser Phe 2O
<210s, SEQ ID NO 185 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine US 2010/0159477 A1 Jun. 24, 2010 77
- Continued <4 OOs, SEQUENCE: 185 Ile Cys His Glin Ile Glu Tyr Tyr Phe Gly Asp Phe Asn Lieu Pro Arg 1. 5 1O 15
<210s, SEQ ID NO 186 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 186 Glu Gly Met Asin Pro Ser Tyr Asp Glu Tyr Ala Asp Ser Asp Glu Asp 1. 5 1O 15 Glin His Asp Ala Tyr Lieu. Glu Arg 2O
<210s, SEQ ID NO 187 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 187 Arg Asn Lieu. Glin Asp Asn. Phe Glin Glu Asp Pro Ile Gln Met Ser Met 1. 5 1O 15 Ile Ile Tyr Ser Cys Lieu Lys Glu Glu Arg 2O 25
<210s, SEQ ID NO 188 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 188 Tyr Lieu. Tyr Pro Asn. Ile Asp Lys Asp His Ala Phe Gly Lys 1. 5 1O
<210s, SEQ ID NO 189 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 189 Glin Phe Lieu Ala Pro Trp Ile Glu Ser Glin Asp Trp Ala Tyr Ala Ala 1. 5 1O 15 Ser Lys
<210s, SEQ ID NO 190 &211s LENGTH: 27 US 2010/0159477 A1 Jun. 24, 2010 78
- Continued
212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 190 Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr Lieu. Tyr 1. 5 1O 15 Pro Asp Ile Pro Llys Glu Glu Ala Phe Gly Lys 2O 25
<210s, SEQ ID NO 191 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 191 Tyr Cys Arg Pro Glu Ser Glin Glu. His Pro Glu Ala Asp Pro Gly Ser 1. 5 1O 15 Ala Ala Pro Tyr Lieu Lys 2O
<210s, SEQ ID NO 192 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 192 Lys Lieu. Glin Gln Thr Glin Glu Tyr Phe Ile Ile Glin Tyr Glin Glu Ser 1. 5 1O 15 Lieu. Arg
<210s, SEQ ID NO 193 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 193 Tyr Tyr Thr Pro Val Pro Cys Glu Ser Ala Thr Ala Lys 1. 5 1O
<210s, SEQ ID NO 194 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 194 US 2010/0159477 A1 Jun. 24, 2010 79
- Continued
Tyr Ala Lieu Ala Val Arg Glin Asp Val Ile Asn. Thir Lieu. Lieu Pro Llys 1. 5 1O 15
<210s, SEQ ID NO 195 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 195 Arg Ala Glu Ala Lieu. Glin Ser Ile Ala Thr Asp Pro Gly Lieu. Tyr Glin 1. 5 1O 15
Met Leu Pro Arg 2O
<210s, SEQ ID NO 196 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (24) . . (24) <223> OTHER INFORMATION: Phosphorylated Tyrosine < 4 OO > SEQUENCE: 196 Asp Gly Asp Thr Glin Thr Asp Ala Gly Gly Glu Pro Asp Ser Lieu. Gly 1. 5 1O 15 Glin Glin Pro Thr Asp Thr Pro Tyr Glu Trp Asp Lieu. Asp Llys Llys 2O 25 3O
<210s, SEQ ID NO 197 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OO > SEQUENCE: 197 Glu Phe Asp Glu Asp Ser Asp Glu Lys Glu Glu Glu Glu Asp Thr Tyr 1. 5 1O 15 Glu Lys
<210s, SEQ ID NO 198 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 198 Val Phe Asp Asp Glu Ser Asp Glu Lys Glu Asp Glu Glu Tyr Ala Asp 1. 5 1O 15 Glu Lys
<210s, SEQ ID NO 199 US 2010/0159477 A1 Jun. 24, 2010 80
- Continued
&211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 199 Thr Pro Asp Gly Lys Val Tyr Tyr Tyr Asn Ala Arg Thr Arg 1. 5 1O
<210s, SEQ ID NO 2 OO &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2OO Thr Pro Asp Gly Lys Val Tyr Tyr Tyr Asn Ala Arg Thr Arg 1. 5 1O
<210s, SEQ ID NO 2 O1 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O1 Gly Phe Thr Asp Ser Pro His Tyr Ser Asp His Lieu. Asn Asp Ser Arg 1. 5 1O 15
<210s, SEQ ID NO 202 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O2 Glin Asp Lieu. Gly Lieu. Gly Ser Pro Ala Glin Lieu. Ser Ser Ser Gly Lys 1. 5 1O 15 Pro Gly Thr Ala Tyr Tyr Ser Phe Ser Ala Thr Ser Ser Arg 2O 25 3O
<210s, SEQ ID NO 203 &211s LENGTH: 30 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O3 Glin Asp Lieu. Gly Lieu. Gly Ser Pro Ala Glin Lieu. Ser Ser Ser Gly Lys 1. 5 1O 15 US 2010/0159477 A1 Jun. 24, 2010 81
- Continued Pro Gly Thr Ala Tyr Tyr Ser Phe Ser Ala Thr Ser Ser Arg 2O 25 3O
<210s, SEQ ID NO 204 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 204 Glin Ile Asn Lieu. Thr Asp Tyr Pro Ile Pro Arg 1. 5 1O
<210s, SEQ ID NO 205 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 205 Glin Val Val Glu Lys Val Glu Thr Glu Tyr Ala Arg Tyr Glu Asn Gly 1. 5 1O 15 His Tyr Ser Tyr Arg Ile His Arg 2O
<210s, SEQ ID NO 206 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O6 Glin Val Val Glu Lys Val Glu Thr Glu Tyr Ala Arg Tyr Glu Asn Gly 1. 5 1O 15 His Tyr Ser Tyr Arg Ile His Arg 2O
<210s, SEQ ID NO 2 O7 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 2O7 Glin Val Val Glu Lys Val Glu Thr Glu Tyr Ala Arg Tyr Glu Asn Gly 1. 5 1O 15 His Tyr Ser Tyr Arg Ile His Arg 2O
<210s, SEQ ID NO 208 &211s LENGTH: 16 212. TYPE: PRT US 2010/0159477 A1 Jun. 24, 2010 82
- Continued <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 208 Lieu. Glin Pro Ile Tyr Trp Ser Arg Asp Asp Wall Ala Glin Trp Lieu Lys 1. 5 1O 15
<210s, SEQ ID NO 209 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 209 Tyr Arg Ser Pro His Ser Gly Asp Val Lieu. Tyr Glu Lieu. Lieu. Glin His 1. 5 1O 15 Ile Lieu Lys
<210s, SEQ ID NO 210 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 210 Lieu. Lieu. Trp Asp Tyr Val Tyr Glin Lieu. Lieu. Ser Asp Ser Arg 1. 5 1O
<210s, SEQ ID NO 211 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 211 His Tyr Tyr Lys Lieu. Asn. Ile Ile Arg 1. 5
<210s, SEQ ID NO 212 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Phosphorylated Tyrosine
<4 OOs, SEQUENCE: 212 His Tyr Tyr Lys Lieu. Asn. Ile Ile Arg 1. 5
<210s, SEQ ID NO 213 &211s LENGTH: 13