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