Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry.
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.
Center for Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160, USA.
Eugene
E
Ciccimaro
Steven K
SK
Hanks
Kenneth H
KH
Yu
Ian A
IA
Blair
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D051416:Focal Adhesion Protein-Tyrosine Kinases; D014357:Trypsin