Identification of Protein Networks Associated with the PAK1−βPIX−GIT1−Paxillin Signaling Complex by Mass Spectrometry

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Abstract
The process of cell motility involves coordinate signaling events among proteins associated in interactive integrin-linked networks. Mass spectrometric analysis of immunoprecipitation-derived protein mixtures have provided efficient means of identifying proteomes. In this study, we investigate strategies to enhance the detection of interactome proteins for the known signaling module: PAK1, βPIX, GIT1, and paxillin. Our results indicate that near-endogenous expression levels of bait protein enhances the identification of associated proteins, and that phosphatase inhibition augments the detection of specific protein interactions. Following the analysis of a large pool of spectral data, we have identified and mapped clusters of proteins that either share common interactions among the four bait proteins of interest or are exclusive to single bait proteins. Taken together, these data indicate that biochemical manipulations can enhance the ability for LC−MS/MS to identify interactome proteins, and that qualitative screening of multiple samples leads to the compilation of proteins associated with a known plexus. Keywords: PAK • PIX • GIT • paxillin • proteomics • mass spectrometry • MS/MS • cell migration • binding partners