Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry.
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.
Schroeder
Melanie J
MJ
Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904, USA. mjschroe@uchicago.edu
Webb
Donna J
DJ
Shabanowitz
Jeffrey
J
Horwitz
Alan F
AF
Hunt
Donald F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004583:Electrons; D022001:Focal Adhesions; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D051419:Paxillin / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D014638:Vanadates / Q000737:chemistry
D051419:Paxillin; D010455:Peptides; C069276:peroxovanadate; D049452:Green Fluorescent Proteins; D014638:Vanadates; D012694:Serine; D014357:Trypsin