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    J Proteome Res. 2005 Sep-Oct;4(5):1832-41.

    Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry.

    Schroeder MJ, Webb DJ, Shabanowitz J, Horwitz AF, Hunt DF.

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    Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904, USA. mjschroe@uchicago.edu

    Abstract

    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.

    PMID:
    16212439
    [PubMed - indexed for MEDLINE]

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