10 20 30 40 50 60 70 80 90 100
GIT1_HUM msrkgprAEVCADCSAPDPGWASISRGVLVCDECCSVHRslGRHISIVKHLrHSAWPPTLLQMVHTLASNGANSIWEHSLLDPAQVQSGRRKANPQDKVH
All Expts
IMAC_INHIB
110 120 130 140 150 160 170 180 190 200
GIT1_HUM PIKSEFIRAKYQMLAFVHKLPCRDDDGVTAKdlskqlhssvrTGNLETCLRLLSLGAQANFFHPEKGTTPLHVAAKAGQTLQAELLVVYGADPGSPDVNG
All Expts
IMAC_INHIB
210 220 230 240 250 260 270 280 290 300
GIT1_HUM RTPIDYARQAGHHELAERLVECQYELTDRLAFYlcgrKPDHKNGHYIIPQMADRsrQKCMSQSLDLSELAKaakKKLQALSNRLFEELAMDVYDEVDRRE
All Expts * * *
IMAC_INHIB * *
NGHYIIPQMADR
LFEELAMDVYDEVDRR
310 320 330 340 350 360 370 380 390 400
GIT1_HUM NDAVWLATQNHSTLVTERSAVPFLPVNPEYSATRnqgrqklarfnarEFATLIIDILSEAKRrqqgkSLSSPTDNLELSLRSQSDLDDQHDYDSVASDED
All Expts [* * ? [ * * * *
IMAC_INHIB * [ * * * *
SEAKRRQQGKSLSSPTDNLEL
SQSDLDDQHDYDSVASDED
410 420 430 440 450 460 470 480 490 500
GIT1_HUM TDQEPLRstgatrsnrARSMDSSDVSDGAVTLQEYLELKKALATSEAKVQQLMKVNSSLSDELRRLQREIHKLQAKNLQLRQPPGPVPTPPLPSERAEHT
All Expts * * [] ] *
IMAC_INHIB * * [] [] *
ARSMDSSDVSDGAVTLQEYLELKK QPPGPVPTPPLPSER
TDQEPLR KVNSSLSDEL AEHT
510 520 530 540 550 560 570 580 590 600
GIT1_HUM PMAPGGSTHRrDRQAFSMYEPGSALKPFGGPPGDELTTRLQPFHSTELEDDAIYSVHVPAGLYRIRKGVSASAVPFtpssplpscsqegsrHTSKLSRHG
All Expts [] [ * *] ** * * [ ]
IMAC_INHIB [] * * *] * *
QAFSMYEPGSALKPFGGPPGDELTTR IRKGVSASAVPF
PMAPGGSTHR LQPFHSTELEDDAIYSVHVPAGLYR HG
610 620 630 640 650 660 670 680 690 700
GIT1_HUM SGADSDYENTQSGDPLLGLEGKrFLELGKEEDFHPELESLDGDLDPGLPSTEDVILKTEQVTKNIQELLRAAQEFKHDSFVPCSEKIHLAVTEMASLFPK
All Expts * * * * *
IMAC_INHIB * * *
SGADSDYENTQSGDPLLGLEGKR
710 720 730 740 750 760 770 780
GIT1_HUM RPALEPVRSSLRLLNASAYRlqsecrKTVPPEPGAPVDFQLLTQQVIQCAYDIAKaakQLVTITTRekKQSTDYKDDDDK
All Expts [] *
IMAC_INHIB [] *
RPALEPVRSSLR
KQSTDYKDDDDK
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. Generally, 10X more peptide than was used for RP separation was converted to methyl esters. Phosphopeptide methyl esters were enriched by IMAC followed by reverse phase separation of peptides over a 1 hr gradient coupled with online detection of phosphopeptides by ESI tandem mass spectrometry (Thermo Electron, LCQ XP).