US 20100151483A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0151483 A1 Hornbeck et al. (43) Pub. Date: Jun. 17, 2010 (54) REAGENTS FOR THE DETECTION OF Related U.S. Application Data PROTEIN PHOSPHORYLATION IN (60) Provisional application No. 60/830,549, filed on Jul. SIGNALNG PATHWAYS 13, 2006. (75) Inventors: Peter Hornbeck, Magnolia, MA Publication Classification (US); Valerie Goss, Seabrook, NH (51) Int. Cl. (US); Kimberly Lee, Seattle, WA GOIN 33/53 (2006.01) (US); Ting-Lei Gu, Woburn, MA C07K 6/8 (2006.01) (US); Albrecht Moritz, Salem, MA (52) U.S. Cl. ....................................... 435/7.1:530/387.9 (US) (57) ABSTRACT Correspondence Address: The invention discloses novel phosphorylation sites identi Nancy Chiu Wilker, Ph.D. fied in signal transduction proteins and pathways, and pro Chief Intellectual Property Counsel vides phosphorylation-site specific antibodies and heavy-iso CELL SIGNALING TECHNOLOGY, INC., 3 tope labeled peptides (AQUA peptides) for the selective Trask Lane detection and quantification of these phosphorylated sites/ Danvers, MA 01923 (US) proteins, as well as methods of using the reagents for Such purpose. Among the phosphorylation sites identified are sites (73) Assignee: CELL SIGNALNG occurring in the following protein types: adaptor/scaffold TECHNOLOGY, INC., Danvers, proteins, adhesion/extracellular matrix protein, apoptosis MA (US) proteins, calcium binding proteins, cell cycle regulation pro teins, chaperone proteins, chromatin, DNA binding/repair/ (21) Appl. No.: 12/309,312 replication proteins, cytoskeletal proteins, endoplasmic reticulum or golgi proteins, enzyme proteins, G/regulator (22) PCT Filed: Jul. 13, 2007 proteins, inhibitor proteins, motor/contractile proteins, phos phatase, protease, Ser/Thr protein kinases, protein kinase PCT/US2007/073534 (Tyr)s, receptor/channel/cell surface proteins, RNA binding (86). PCT No.: proteins, transcriptional regulators, tumor suppressor pro S371 (c)(1), teins, ubiquitan conjugating system proteins and proteins of (2), (4) Date: Feb. 19, 2010 unknown function. Patent Application Publication Jun. 17, 2010 Sheet 1 of 22 US 2010/0151483 A1 FIGURE 1 crude protein (e.g., cell extractor cellular organelle) or invirokinase reaction or purified phosphoprotein obtain protein faction (e.g., lysis, organic extraction, detergent treatment, etc.) immobilize general protein modification-specific antibody to resin denature proteins (e.g., heat treatment) digest crude protein mixture to peptides withimmobilized trypsin or other protease contact digest with antibody-resin and incubate (e.g., 4, 1-16 hours) remove unbound peptides by centrifugation, filtration, or column washing 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) desalt and concetrate peptides withmicrocolumn (e.g., reversed-phase ZipTip) (optional) analyze, e.g., MS for mass, MS after phosphatase treatement to confirm phosphorylation, or tandem MS (MS/MS or MS) for partial sequence Patent Application Publication Jun. 17, 2010 Sheet 3 of 22 US 2010/0151483 A1 Figure 2 Celine Ng. Protein Type Phosphorylation Site Sequence Disease Tissue Patien ------- Adhesion Cr extracellular T cell NP-001085090.1 matrix protein DyEPPSPSPAPGAPPPPPOR leukerTia Jurkat BceAL VEQCPDyr ------------------------------mi-n-n NP158852 imatic protein MSEPPWCNL ----- Gel All Adhesion or extracellular NP_0362042 AMENOySPTPGTDC NP 906557.4 matic protein SPSTGOPTNQSMDDTREDIYNNPTFSR Adhesion or extracellular NP-019.72 mali protein DSSCGTGyELTEDNSCs lymphoma transform NP-060217. LEGEWTPNSLSTSyk edkidney NPOO4313. SRSAPPNLWAAQRyGRELRR CML AML NP-004231 Y139 SHLMSLySACSSEVPHGPWDOK T celAll MOTIS - AML Tcel Jurkat NP_004865.1 Y102 leukemia MO-91 T cel Yi SGYGPSDGPSyGR leukemia Jurkat Tce NP_004865, SVPCSGPTVRPCEDAWASPGAyGMGGR leukemia lurkat cell NP_004865. Y302 leukemia Jurkat Teell NP-004365. SGyGPSDGPSYGR leukemia Jurkat KG-1 MKPL.1 NPCO1743. GAGAFGFEWTHDITK AML Monomac 6 NPC55939. CTW-1 AM OLT15 NP_00148, Calcium-binding proten TPWLFDlyEIKEAIK T cell ALL VAL CTW.1 AML H1869 NSCLC HCC1806 Tcell AL MOLT15 breast NCI-N87 cancer R gastric csO01 NP 001148. Calcium-binding protein DAQELyAAGENR cancer csO42 AML CW NP-004030, Y199 DYAGWKR TceALMCLT15 AML CTV-1 NP001145.1 Calcium-binding proten ... Y255 SIPAYLAETLYYAMK Tce AL MOLT15 NP_001145.1 Calcium-binding protein Y297 NFATSLySMIK AMt CW-1 NP-004024.2 NKPLFFADKyk T cell ALL MOLT15 NP004024.2 CalciuT-binding protein LINGLMRPPAYCDAK lymphoma SUPT-13 acute erythrobla stic NP-001731.1 calcium-binding protein SGyIDEHELOALLKDLYEK leukemia HEL 0.04334.1 ng protein I FyALSASFEPFSNK DLBC Pfeifer CTW NP005436.1 Cell cycle regulation hTEEEKEELAOyakWDK AM MKPL-1 NP.005436. Cell cycle regulation CML K562 83-102F NP005436. Cell cycle regulation GALTGGYDTRK NP-00476, Cell cycle regulation ACL S NP0476, Cell cycle regulation WAVEyLDPSPEVOK AM. KG. NP-0046522 Cell cycle regulation ALCL. IIS Tell NP-0574273 Cell cycle regulation LEyFSCDHOELLOR lekema Jurka cell NP0556253 Cell cycle regulator SHIPENFDDVDINEDEDCYSDER leukemia Jurkat Tct NP-955625.3 Ceccle reguation SHPENFDCYWOINEDOCSDER leukemia Jurkat Tcel NP-0556.253. Cell cycle regulation leukemia Jurkat Tce) NP 071394.2 Cell cycle regulation NCyCALKPR leukemia Jurkat EaF3.0ZF A. BaF34ZF NP_003531.1 Cell cycle regulation CML CHRF AML CTW.1 NF-06420.1 Chaperone. WHTVEDYQAMDAEWNLOKLEk Tcell ALL MOLT5 NP 0753.1 ChaperOne KRVEDAyTCNWSLEYEK ALCL. TS Patent Application Publication Jun. 17, 2010 Sheet 10 of 22 US 2010/0151483 A1 Figure 2 Phospho. Celine Protein Name Accession No. Protein Type Residue Phosphorylation Sita Sequence Disease Tissue Patient KG KG1-A Receptor, channel, transporter AML MO-91 ATPSH NP 008347.1 ...arcel surface Protein-YSE LAALPENPPADWAYYK T ce. AllMOLT15 Receptor, channel, transporter ATP6VA NP-001884.2 or cell surface protein IY485 ALDEYyDKHFTEFVPLR CMBaF3PRTK Receptor, channel, transporter ATP6VC NP-00186.1 or ceasurface protein Y367 HLDSSAAACAPMOPGNLSQQYyPYWYYK CMLBaF3-10ZF Receptor, channel, transporter ATP6VE NP_001687.1 or cell surface protein Y56 IMEyYEK. ---- CMLBaF3-4ZF Receptor, channel, transporter TS ATP6VH legius orcell surface protein Y388 LNEKNyELK ALCL licsO15 Receptor, channel, transporter ATP8B NP-005594.1 or cell surface protein ... Y1217 GyADLISSGR SCLCDMS 53 Receptor, channel, transporter T cell NP-01694.2 or cell surface protein Y655 AVPSADDVQR - leukemia Jurkat Receptor, channel, transporter - NF-008925.1 or celsurface protein Y295 EREMKEMGyAATEGESLREKLQEELK DLBCL OC-ly GDM f HU-3 KG-1 : KG1-A Receptor, channel, transporter Me-F2 -- NP_001020280.1 or cell surface protein Y330 LGEDPYTENGGGOGYSSGPGTSPEAQGK AML hit-44b OND4 ALL GDM-1 AML Jurkat Receptor, channel, transporter T cell KG1-A NP906128.1 or cell surface rotein Y222 DKNSAACWEDMSHSR leukemia lung tumor T57 Receptor, channel, transporter NP-003865.1 or cell surface protein Y264 TYTylMASR ----------------- --- AMLICHRF AML acute CHRF erythrobla ELF-153 Receptor, channel, transporter stic HEL NP-003865.1 or cell surface grotein Y324 ASTQDSKPPGTSSyEV leukemia gM -1 RC-KS Werona 25S gz68 gzB1 Receptor, channel, transporter ALCL lung tumor T57 - NP-001868.2 or cell surface protein Y1029 WSWDPyNPAS AML patient 6 Receptor, channel, transporter Tell eIF4ENIF NP-062817.1 of cell surface protein Y580 AASADyLRPR -1. Jurkat Receptor, channel, transporter multiple NP-000061 or cell surface protein Y436 FKANDHGONER myelona. KMSB Receptor, channel, transporter -- NP-112571.1 or censurface protein Y924 GENWSEVR AML 255 T cell CPSF6 NP-008938, RNA binding protein Y364 GFPPTDPyGRPPPYDR -m- leukemiarkat AML Tce Jurkat PSF6 . NP-0089381 RNAbinding protein. Y74 GAAPNWTTGKR ... .leukemia. MKPL-1 stF-50 NP_001315.1 RNAbinging protein Y195 TLYDNDEVTCUAFHPTECILASGSRDyTK ALCl. TS . Tce? CsIF-50 NP-001315.1 |Rubids Protein. Y6 LGMENDDTAVCyAGR, ... ........ leukemia Jurkat CsIF77. NPOO13171 RNAbinging protein Y534 AMLKG-1 AM CTN-1 CUGBP2 NP-006S$2.3 RNA binding protein Y70 ELFEPyGAWONWR Tel AL MOLT15 NP C0407.2.2 RNA indigarotein Y515 OAFPAYPSSPFONTTGy(LPVYNY. ...Cl 223. CL NP C0407.22 RNA binding protein Y520 (AFPAYPSSPFCWTTGYOLPWNY CL 223. C. DA2 NP_0040722 RNA binding protein Y522 IQAFPAYPSSPFONTTGYOPWNy E. 223. Cll NP-0043092 RNA binding protein Y470 DRIEESDQGPyAILAPTR -- ALCLTS Post: RNAbinding protein Y30 WRPCWYGGADIGQQR K562 NP-0013473. RNAbinding protein. Y53 GEyOKDSSGWSSSKDKDAYSSFGSR leukemia Jurkat DDX5 NP-004387, RNAbinding protein Y58 E. - AML MKPL.1 NP-004387.1 RNAbinding protein Y97 GHNCPKPWLNFyEANFPANYMDVIAR Tcell ALL MOTS Act KG-1 NP-003541RNAbinding protein Y153 SQQSACKEYWGVR . - ... AML TS 303 BAP5 NF-00897.2 RNAbinding protein Y510 NYLDOTNWyGSAORR Tel ALL MOLT15 EAVL1 NP-0014102 RNAbinding protein Y109 WSYARPSSVKDANLySGLPR AM MKPL-1 CML Tce Arkat Al NP_0603261 RNA binding protein Y79 CSYWNADHDyCNR leukemia K562 Tcel - |NF-005548.1. Transcriptioral regulator. Y522 MLSTSEySOSPK . leukemia Jurkat T cell --- leopa Transcriptional regulator Y64 ONMLGNyEEWK leukemia Jurkat Tce NP-003968. 1 Transcriptionalegulator Y247 WEEyYEWLDHCSSINK leukemia Jurkat - KG-1 NP-1103942. Transcriptional regulator Y356 GQLNyPLPDFSK AML KG-A NPOO6319.2 Transcriptional regulator Y375 ToyFSSTD6, Tcell AL MOTS Patent Application Publication Jun. 17, 2010 Sheet 11 of 22 US 2010/0151483 A1 Figure 2 B w F G Phospho- Celine 1 Protein Name Accession No. Protein Type Residue Phosphorylation Site Sequence Disease issus Patien -- - - - - - - - - - - - BaF3. AML FLT3(D842Y) NSCLC