US 20100129929A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0129929 A1 Polakewicz et al. (43) Pub. Date: May 27, 2010 (54) TYROSINE PHOSPHORYLATION SITES (86). PCT No.: PCT/US2007/O16927 (76) Inventors: Roberto Polakewicz, Lexington, S371 (c)(1), MA (US); Charles Farnsworth, (2), (4) Date: Feb. 13, 2010 Concord, MA (US); Ailan Guo, Related U.S. Application Data Burlington, MA (US); Klarisa Rikova, Reading, MA (US); (60) Provisional application No. 60/833,826, filed on Jul. Albrecht Moritz, Salem, MA (US); 27, 2006. Kimberly Lee, Seattle, WA (US); O O Erik Spek, Cambridge, MA (US) Publication Classification (51) Int. Cl. Correspondence Address: GOIN 33/53 (2006.01) Nancy Chiu Wilker, Ph.D. C07K 6/8 (2006.01) Chief Intellectual Property Counsel (52) U.S. Cl. ...................................... 436/501; 530/387.9 CELL SIGNALING TECHNOLOGY, INC., 3 Trask Lane (57) ABSTRACT Danvers, MA 01923 (US) The invention discloses 349 novel phosphorylation sites iden tified in carcinoma, peptides (including AQUA peptides) (21) Appl. No.: 12/309,727 comprising a phosphorylation site of the invention, antibod ies specifically bind to a novel phosphorylation site of the (22) PCT Filed: Jul. 27, 2007 invention, and diagnostic and therapeutic uses of the above. Patent Application Publication May 27, 2010 Sheet 1 of 22 US 2010/0129929 A1 FIGURE 1 Crude protein (e.g., cell extractor cellular organelle) or in vitrokinase reaction or purified phosphoprotein obtain protein fraction (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 with immobilized 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 w 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 with microcolumn (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 May 27, 2010 Sheet 2 of 22 US 2010/0129929 A1 Figure 2 Protein . Accessie Phosphe. a lirassis isie No. Protein ype Residue Phosphorylation Site Sequence Disease airs S Aiii. NP_001611.1 Adaptofiscaffold Y8S vKGEyDWTMPK isfiasts 33 S. iii. is: fix P 3503.2 Adap::iscaffod 33 FOCTYPSGGEEACCR go aire SW620 SE. O. 2. Åir N 0403.2 Adagirisatil i. CEC-IEVO Gwysey K ui rice it: a bifas. Si 3. casessi: 3: Cixi NP 55227. Adapteriscified s KyEDGEODMKR cio &ce vis (a-i. NP C06394.1 Adapteriscaffei: y: TGHGYWYEPSR S. 8S (Eastors Squaits ce. G-GS as- NPOS334. Acaptiscarcic f Swhiy Ywi (KEEFER 8::: 33; S.C. far breast Caice, 3. NP {5,343.2 Adaptofiscaffold y MTSDAvy K &as case fee s NSCC Cat-3 cks NF 060589.2 Adaptoriscaffold Y23 GIEiySYPC PT, MPR Cogs 3-cef ES N-03855, Atiatoriscaicis Y148 ALEvy VPPAR - reast casses HCC423 enopaki: N 979, Asia:Criscaffold Y7s2 WOCAA, OFK tung cancer 5. iS3 siycipiiri NP 10:379, Adapteriscaicii Yt703 is EP KDMSPYEAyKR S. s ps3 PO4.433.3 Adaptatisaig 232 Cy-FQEETPE, AAR S -82 S. 323s 3.298 a 75767.2 Adaptofiscaicii y SOESGWEyFSK gastric cancer Cis ischest of exigea : f 574332 matrix protei; Y844 yYPMPFFDELK glioblastefa M059. SEQ) NO. 4 GAG iS X 3. 38 i. isis, KSE2 is C-87 ..? C. liSC s: gastric 3 icef s: giliastina scs oria ga4 Agasif exaces: iting carcer 3. AA POS 438.2 raisix picts: 83 YPyPPPLPDEDKK saia case; is i? 2. S. 15 Achest r exaceria: six MPC592.2 matrix proteir 3. YSyEVHVSGER NSC is8S SE is 16 Aches: extrace.3 aia iOS 2.2 at ti Y;i SEGEy. STONGSPESPOPNAEYSEPK coor cancer 8,82 Si. 17 Asif : 3:3:3: 3 hip 07842 at rotein: y DNVFYyGEEGGGEEDCOYD, OR 333 as: iC3S SiO, 18 Aires: , 8xacif giadii. 3 EP (, 531 atrix riter y: Kiy DGGARTEDEVGSYPSKHY, horia iuri S3 SEQE: NO. 49 Aisic; exactiis C. N 0895. Patrix protein Yi YiyNGASWA SSR irriga; Cer 3. SEO NO2 Aasif acaf Caf2 N° 0183.2 matrix protein Y3. CyPGESGEKGSGCKSF gicia: A172 Sii 2. Adhesis; taxatieta -25s Chis *: O(85.1 matrix proteir 3. AAVSHWQQQSy DSG HSGATTAPSLSGK St. CC82 S. O ki is a cer sts Aciesis fatalia: Siasis : iS; sis NP 08:3, matrix protein 8 HyGPWvK 3::::::::: sji: S. Aless crexasia: is a reas case Patent Application Publication May 27, 2010 Sheet 3 of 22 US 2010/0129929 A1 Figure 2 f 3. s Prote Access Phospho Celtic isstei liaise is, Protein ype Reside Phosphoryliatic Site Segence sease arist SEO Aches of extraceliar i?ian reas cance: : CXA NOC329 patrix fairs SYGSNHSSGSMSPSNAEGySK if is acs: Cirie SEO iO2S: fides of extraces: 8. s C-S PO3728.1 matrix ceil LDREERDAyNLR east are: i. S. iii. Acision 3 extrasiliar f:38 cas: CC8 8 sissistakin if CG44.08.2 matrix protein SVEORFDOCKhiy OQLOK coincarcif Si62 SEQDK.C. 27 ises axiacetitisf esota Fe O2 atrix rosei SykDANRSAVEDITG R S. six SEQD NO. 28. Aces of f extracelia 3. S. N 777 AC. matrix proteir YS WYLCNQDENHKHAQDywi YNYEGR CC8 SE 29 Assis a sex acetia S. N 07774.1 matrix protein SCC 32? SEQD No. 30 3. CASP8AF2 NF 3634 Assissis NCNSDyCGSEGMEMK SC Cat-3 SEC E iO. 3 gastric cancer 3. t axia NPOC43.1 i-dig Rite: Yi PAQy DASEK gic tastora C-87 SEOD NO 32 ski, NP C04030, F-ixir; ifosi YS AFLNVOCONKPYFADRy Ski K gastric career C-87 SEO. O. 33 C2 NO38259.2 Cecycle regulation Y8. EvKGSES-AYGwww.GYCR Sea SEQD NO. 34 C NP 036259.2 Ceil cycle regulation Y 38 EWKGSETYSPNTayGWOFWAASYCR N. S. Cel cycle regiation y EWKGSEYSPAYGVFAP GyCR - - SEOSENO NC 3.39 -8 C isit S. ico carcer Siii.5 gastic catcar XG . 3. class ;P 71394.2 Cast cycle regain ESA. Ly-Eik See X2 SEQID NO. 37 3A 3. Cl2 NP35823 Cai cycle regula-Gi My SJSALNNK tung carcef s: SEQ O NO. 38 3. arets 3 ip633483. Chapeities FyASARFKPFS-K gas,Caitei --8 SEQD NO 39 Chromain, DNA-binding DNA repair of NA : 3. NP 00:48.3 regication rosei y rostate acer SEC DNO. 40 Croatin, Dia-bindirig, A regar of NA Bright NC 525 replication protein CVLDLFMywVTEK is six SEC D. No. 4 3ctii, alia NP 009. Cytoskeietal protei TGIVESGGV NWYEGALP-AMR SCC 5X. SEQDNO. 42, al 328 s ;SC. at a...ces fe: iC; NPO1C33, Cytoskeleia protein X-Phili IAFC AEKypix ce. A iOS SEQ to No. 43. S A. NPO1631. Cytoskeletal proteir SLAACDEGGPELEPDyGTATR 3. 8s: 2. SEOD NO. 44 is capoir 3 NF (33). Cytoskeieia protein CYCySDQG: OY seats as: .A.A-SS SEC DNC is C a feast career sis east 3.8F NC-8 O42.2 Cytoskelia ite LASy. KiR gastric sée: 22 DNC 46 0413, Cytoskelstartei EGEDAF-LTOK gast 10 NO.47 iP25. Cytoskeletaliotein STFSTNyR ci. is 3. S. iii. 8 kiPOCO25, Cytoskeletal fier Y255 AOy AR gastric aficer C. S C ; reas:cancer CC86 AAA33A, Cytoskeleta iteir YS ySSSSSGGGGGYGGAES GAGNEK assassi Ci-- S. NSCC (als-3 Cix: AAA35044. Cytoskeletal protein SS FVSSSSSGGYGGGyGGv.IASDG. AGNEK gastric carcer is SE x-E CK13 NFCO228.2 Cytoskeletarie DYSHYYTIGER. gastric cance... gzi St. i. Patent Application Publication May 27, 2010 Sheet 4 of 22 US 2010/0129929 A1 Figure 2 ri Accessic Phospho Celine fissisi is: . Proteiype Reside rosphorylation Site Sequence Disease aii SEC SCL feast acci S. gastric carcer iC8 status ce. iCC827 3. NP (2267.2. Cytoskeleta frien Y256 SCEVRAEONRK acic C-8 SECD No. 53 C3 NP C02237,2:Cytoskeleiai ser Y8. SRLEQEATyr asic cances C-87 SEC D. No 54 CC cs: treas:(3.8; g22 C Pi3.2 Cytoskel:aise wSLAGACGGGYGSRSyNGGSKR Colo? cance? s4. SEC his C. P.O.S, Cytoskeletal protein 283 CVCAA vitive AK ling cancer ga SE is | gait is is is 0452 Cytoskeiseia its Y3A AOyEEANR as 3. iting tumor 26 S. N. S. 8:8 carce; 22 NPO452 Cytoskeleta protein Y. SRTEAESWyCTKYEELCQTAGR coccaces sit SEC - 8 C38 NP (45.2 Cytoskeletarifier 3. SRTEAESNY OTKOOTAR east case: gE S. O. SS kia, is case: ca NP775103, Cytoskeisa protein 32 Sly GGGSKR 3s cate gii SE : . trait seast carcer at is NP 77 109.1 Cytoskeietal protein Y3. ISIGGGSCAISGSyGSR feast acer ge: S NSCO NPOS473 Cytoskeleta protein YSS SAyGGPVGAGR gastric cance ii. 3 SEQ DNQ 62 888 34 its CC13, i-CC S. *:C-87 gastric arcer s if OOSSAT 3 Cytoskeleta protein y (DVOAA SK prostate cancer assi SE 3 8. iPO06543 Cytoskeleta prote: 283 AEAEAWyCIKFECACAGK ESCC CC8 SEC x; }; NP 002264. 1 Cytoskeletal protein 2S SySGPGSR i? cance Ciá3. Six, ; iO236, Cytoskeleta prote: ix.EAELGNOGLEDFKNKyCENKR gastic career NC-N87 Sii, S. is & seascace 23 C: Cicer C3 s gas, it cance: Ci-- Safius ce. via 3 NP_0022641. Cytoskeletal protein YS SRAEAESMyCKYFLOSLAGK carcino: Sé8. ice ii. is N22EA, Cytoskeleta prote: SS SRAEAESMYCKyEES AGK gastic carcer AC32 Cat-3 S.C. C-87 is NPS5:17.1 Cytoskeleta protein YS SNYyCAYOAOPATR gastric C3: Ce: s: C-3 Caci, 2 (Cytoskeleta rotein Y: SNYYOAyOAQPLATR astric carcer ga ciatic Cytoskeleta Ectein Y. SENSSTOYi cs: cacsi CC:33 is catar 2. Scarious ce. id at 3 NP_001297. 1 Cytoskeletal protein y wwySAR Cicilia 43 SEO NO2 s gastricances A 3 (Otacts NPO3222.2 Cytoskeleta role Yig SAG-Dy(GKEK 3 :: C.E. SEQD No. 73. Ca P (1894.2 Cytoskeleta rotein Y2C, OCA, GSAAAASOASOHOGGGGGENSCC SEQD NO 74 Cia iP834. Cytosieleasotei y NGREEKEW&CyåOVFR NSCLC 5 x SEQDNC 75 SCC Cat-3 s Cixi NP (323.