Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry.

Ciccimaro E, Hanks SK, Yu KH, Blair IA
Anal Chem. 2009 81 (9): 3304-13

PMID: 19354260 · PMCID: PMC2706532 · DOI:10.1021/ac900204f

A vital point of convergence for many signaling pathways at cellular focal adhesions is the interaction of two nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and Src. The binding of Src to FAK leads to the phosphorylation of Y(576) and Y(577), located within the activation loop domain of FAK. However, it has not been possible previously to determine the absolute quantitative relationship between phosphorylated and nonphosphorylated forms of this activation loop domain in cells undergoing normal metabolism. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows such determinations to be made. Isotopically labeled and phosphorylated FAK protein standards were synthesized and used to control for loss during immunoprecipitation of FAK. A control tryptic peptide, representing an unmodified region of FAK, was employed to monitor the mass balance of post-translational modifications (PTMs) on the activation loop domain. Absolute quantification was conducted using stable isotope labeled peptide standards with four endogenous amino acid overhangs at the trypsin digestion sites of both the amino and carboxy terminus. The LC-MRM/MS method was rigorously validated using in vitro kinase assays and employed to conduct absolute quantification of FAK phosphorylation in normal mouse embryonic fibroblasts (MEFs). This methodology will have particular utility for biomarker studies of kinase-inhibiting anticancer drugs and for quantitative proteomic investigations that examine kinase- and phosphatase-mediated cellular signal transduction pathways.

MeSH Terms (13)

Amino Acid Sequence Chromatography, Liquid Enzyme Activation Focal Adhesion Protein-Tyrosine Kinases Humans Immunoprecipitation Isotope Labeling Mass Spectrometry Molecular Sequence Data Peptides Phosphorylation Reproducibility of Results Trypsin

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