PAXI_CHICK mdykddddkgpkgtvskGEELFTGVVPILVELDGDVNGHKfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrYPDHMKQHDFFKS
All Expts
IMAC_INHIB
PAXI_CHICK AMPEGYVQERTIFFKddgnyktraevkFEGDTLVNRielkGIDFKEDGNILGHKleynynshnvyimadkqkngikvnfkirHNIEDGSVQLADHYQQNT
All Expts
IMAC_INHIB
1 10 20 30 40
PAXI_CHICK PIGDGPVLLPDNHYLSTQSALSKdpnekrDHMVLLEFVTAAGITLGMDELYKMDDLDALLADLESTTSHISKRPVFLTEETPYSYPTGNHTYQEIAVPPP
All Expts *
IMAC_INHIB *
RPVFLTEETPYSYPTGN
50 60 70 80 90 100 110 120 130 140
PAXI_CHICK VPPPPSSEALNGTVIDPLDQWQPSVSRYGHQQPQSQSPIYSSSAKSSSASVPRDGLSSPSPRASEEEHVYSFPNKQKSAEPSPTMTSTSLGSNLSELDRL
All Expts * * ** []] * * * * ** * * *
IMAC_INHIB * * ** []] * * * * ** * * *
SSSASVPRDGLSSPSPR SAEPSPTMTSTSLGSNLSELDR
YGHQQPQSQSPIYSSSAK ASEEEHVYSFPNKQK
150 160 170 180 190 200 210 220 230 240
PAXI_CHICK LLELNAVQHNPPSGFSADEVSRSPSLPNVTGPHYVIPESSSSAGGKaapptkekpkrnggrGIEDVRPSVESLLDELESSVPSPVPAITVSQGEVSSPQR
All Expts * * * * [ ] [] * * *
IMAC_INHIB * * * [ ] [] * * *
GIEDVRPSVESLLDELESSVPSPVPAITVSQGEVSSPQR
SPSLPNVTGPHYVIPESSSSAGGK
250 260 270 280 290 300 310 320 330 340
PAXI_CHICK VNASQQQTRISASSATRELDELMASLSDFKFMAQGKAGGSSSPPSTTPKPGSQLDTMLGSLQSDLNKLGVATVAKGVCGACKKPIAGQVVTAMGKTWHPE
All Expts * *
IMAC_INHIB *
ISASSATR
350 360 370 380 390 400 410 420 430 440
PAXI_CHICK HFVCTHCQEEIGSRNFFERDGQPYCEKDYHNLFSPRCYYCNGPILDKVVTALDRTWHPEHFFCAQCGVFFGPEGFHEKdgkaycrKDYFDMFAPKcggca
All Expts *
IMAC_INHIB *
DYHNLFSPR
450 460 470 480 490 500 510 520 530 540
PAXI_CHICK rAILENYISALNTLWHPECFVCRecftpfingsffehdgqpycevhyherRGSLCSGCQKPITGRcitamgkKFHPEHFVCAFCLKqlnkGTFKEQNDKP
All Expts *
IMAC_INHIB *
RGSLCSGCQKPITGR
550
PAXI_CHICK YCQNCFLKlfc
All Expts
IMAC_INHIB
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. |
|
1.5-2e7 HEK cells were transiently transfected with 10 ng FLAG-GFP-Paxillin DNA and 4 ug pBS (empty vector). 1 mM peroxovandate and 10 nM Calyculin A pretreatment of cells was for 30 min prior to lysis. Extraction was for 30 min at 4degC in 25mM Tris, 100 mM NaCl, 0.5% NP-40, 1 mM peroxovanadate, 10 nM Calyculin A. Pre-cleared once with 30 uL non-specific IgG agarose for 1 hr at 4degC. Repeated preclear overnight. Immunoprecipitation (IP) was with 100 uL anti-FLAG IgG agarose beads slurry for 1.5 hr at 4degC. Washed IP 2x with 200 uL 25mM Tris, 100 mM NaCl, pH 7.4 10 min each. 10-15% of the IP was used to visualize purity by silver-stained gel. On-beads digestion of 40% of the IP was with 100 mM ammonium bicarbonate pH 8 and 500 ng trypsin. An aliquot of the digest corresponding to 38% of the original immunoprecipitated paxillin was converted to (d0) methyl esters. Combined with equal amount FLAG-GFP-Paxillin -inhibs (previously converted to d3 methyl esters) for simultaneous enrichment of phosphopeptides 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, LTQ-FTMS).