10 20 30 40 50 60 70 80 90 100
GIT1_HUM msrkgprAEVCADCSAPDPGWASISRGVLVCDECCSVHRslGRHISIVKHLrHSAWPPTLLQMVHTLASNGANSIWEHSLLDPAQVQSGRRKANPQDKVH
All Expts
C18_INHIB
AEVCADCSAPDPGWASISR RHSAWPPTLL KANPQDKVH
GRHISIVKHL TLASNGANSIWEHSLLDPAQVQSGR
110 120 130 140 150 160 170 180 190 200
GIT1_HUM PIKSEFIRAKYQMLAFVHKLPCRDDDGVTAKdlskqlhssvrTGNLETCLRLLSLGAQANFFHPEKGTTPLHVAAKAGQTLQAELLVVYGADPGSPDVNG
All Expts
C18_INHIB
PIK LPCRDDDGVTAK LLSLGAQANFFHPEK AGQTLQAELLVVY
YQMLAF TGNLETCLR GTTPLHVAAK GADPGSPDVNG
210 220 230 240 250 260 270 280 290 300
GIT1_HUM RTPIDYARQAGHHELAERLVECQYELTDRLAFYlcgrKPDHKNGHYIIPQMADRsrQKCMSQSLDLSELAKaakKKLQALSNRLFEELAMDVYDEVDRRE
All Expts * * *
C18_INHIB
ARQAGHHELAERLVECQY NGHYIIPQMADR LFEELAMDVYDEVDRR
RTPIDY ELTDRLAFY SQSLDLSELAKAAK
310 320 330 340 350 360 370 380 390 400
GIT1_HUM NDAVWLATQNHSTLVTERSAVPFLPVNPEYSATRnqgrqklarfnarEFATLIIDILSEAKRrqqgkSLSSPTDNLELSLRSQSDLDDQHDYDSVASDED
All Expts [* * ? [ * * * *
C18_INHIB * * *
SAVPFLPVNPEYSATR SLSSPTDNLELSLR
LATQNHSTLVTER EFATLIIDILSEAKR SQSDLDDQHDYDSVASDED
410 420 430 440 450 460 470 480 490 500
GIT1_HUM TDQEPLRstgatrsnrARSMDSSDVSDGAVTLQEYLELKKALATSEAKVQQLMKVNSSLSDELRRLQREIHKLQAKNLQLRQPPGPVPTPPLPSERAEHT
All Expts * * [] ] *
C18_INHIB *
SNRARSMDSSDVSDGAVTLQEY KVNSSLSDELR QPPGPVPTPPLPSER
TDQEPLR KKALATSEAKVQQLM NLQLR AEHT
510 520 530 540 550 560 570 580 590 600
GIT1_HUM PMAPGGSTHRrDRQAFSMYEPGSALKPFGGPPGDELTTRLQPFHSTELEDDAIYSVHVPAGLYRIRKGVSASAVPFtpssplpscsqegsrHTSKLSRHG
All Expts [] [ * *] ** * * [ ]
C18_INHIB * *
SMYEPGSALKPFGGPPGDELTTR SRHG
PMAPGGSTHR LQPFHSTELEDDAIYSVHVPAGLYR
610 620 630 640 650 660 670 680 690 700
GIT1_HUM SGADSDYENTQSGDPLLGLEGKrFLELGKEEDFHPELESLDGDLDPGLPSTEDVILKTEQVTKNIQELLRAAQEFKHDSFVPCSEKIHLAVTEMASLFPK
All Expts * * * * *
C18_INHIB * * * *
SGADSDYENTQSGDPLLGLEGKR NIQELLR IHLAVTEMASLFPK
FLELGKEEDFHPELESLDGDLDPGLPSTEDVILKTEQVTK HDSFVPCSEK
710 720 730 740 750 760 770 780
GIT1_HUM RPALEPVRSSLRLLNASAYRlqsecrKTVPPEPGAPVDFQLLTQQVIQCAYDIAKaakQLVTITTRekKQSTDYKDDDDK
All Expts [] *
C18_INHIB
RLLNASAY TQQVIQCAY
RPALEPVR KTVPPEPGAPVDFQLL DIAKAAKQL
Legend
| * | Phosphorylated sites are denoted by an asterisk below the amino acid residue. |
| [ ] | The specific phosphorylated amino acid between the brackets can not be distinguished by the MS/MS spectra. |
| All Expts | A summary of all phosphorylation sites detected on this molecule from all experiments. |
| C18_INHIB, C18, IMAC_INHIB, IMAC | Analysis type. |
|
Alternating, aligned peptides represent protein coverage for this experiment
Lower case amino acids have yet to be detected in any enzymatic digest analyzed by mass spectrometry. |
|
1-1.5e7 HEK cells were transiently transfected with 3.5 ug GIT1-FLAG DNA. 1 mM peroxovandate pretreatment (no Calyculin A) of cells for 30 min prior to lysis. Immunoprecipitation (IP) was with 80 uL anti-FLAG IgG agarose beads slurry. 10-15% of the IP was used to visualize purity by silver-stained gel. GIT1 was either eluted with 100 uL of 0.2 mg/mL DYKDDDDK or left on-beads. Eluted GIT1 (corresponding to ca. 8% of the original IP) was reduced with DTT and alkylated with iodoacetamide in 100 mM ammonium bicarbonate and digested with either 500 ng trypsin, chymotrypsin, or a dual trypsin/chymotrypsin digest. Online reverse phase (RP) separation an aliquot of the digest corresponding to ca. 1% of the original immunoprecipitated GIT1 occurred over a 2 hr gradient. On-beads GIT1 (corresponding to 30% of the original IP) was digested with 500 ng trypsin. Online reverse phase (RP) C18 separation of an aliquot of the on-beads digest corresponding to ca. 0.3% of the original immunoprecipitated GIT1 occurred over a 1.5 hr gradient coupled with online detection of phosphopeptides by ESI tandem mass spectrometry (Thermo Electron, LCQ XP).