Journal
JOURNAL OF PROTEOME RESEARCH
Volume 4, Issue 5, Pages 1832-1841Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr0502020
Keywords
paxillin; IMAC; phosphorylation; glycosylation; GlcNAc; methyl esters; peroxovanaclate; calyculin A; ETD
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Funding
- NIGMS NIH HHS [U54 GM 64346, GM 37537] Funding Source: Medline
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Methods for the simultaneous identification of interacting proteins and post-translational modifications of the focal adhesion adapter protein, paxillin, are presented. The strategy includes (1) lower-level, transient transfection of FLAG-GFP-Paxillin into HEK293 cells, (2) incubation of cells with phosphatase inhibitors prior to lysis, (3) purification of paxillin by anti-FLAG immunoprecipitation, (4) analysis of peptides generated from on-beads digestion using LTQ-FT or LTQ-ETD mass spectrometry, and (5) enrichment of phosphopeptide methyl esters with IMAC. Using the above strategies, we identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, with this method, we simultaneously detect 10 co-purifying proteins which are present in focal adhesion complexes. Extension of these methods to other substrates should facilitate generation of global phosphorylation maps and protein-protein interactions for any protein of interest.
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