Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

摘要

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.