Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry

An approach based on nanospray liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) was developed in order to analyze twenty-nine phosphorylated and non-phosphorylated tryptic peptides from focal adhesion kinase (FAK). All peptides monitored were resolved and showed excellent peak shape with the exception of one doubly phosphorylated peptide. Optimization of the LC method enabled the identification and subsequent monitoring of six important tyrosine phosphorylation sites on FAK, including phosphorylated Y-397 (pY(397)), pY(407), pY(576), pY(577), pY(861), and pY(925). This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. FAK was shown to have autocatalytic function, which resulted in efficient phosphorylation of Y-397. FAK was also capable of autophosphorylation on residues Y-407 and Y-576, though apparently less effectively than autophosphorylation at Y-397. Src was found to phosphorylate FAK at Y-407, Y-576, Y-577, and Y-861. The presence of Src increased the abundance of pY(576) at low temperature indicating Src had particularly high kinase activity toward this residue. Furthermore, Src phosphorylated FAK at Y-577 to produce FAK bis-phosphorylated at Y-576 and Y-577. In addition, six novel sites of phosphorylation (Y-148, Y-347, Y-441, T-503, S-850, and Y-1007) were identified on FAK. Interestingly, Src phosphorylated FAK to form a peptide uniquely phosphorylated on Y-407, together with substantial amounts of the bis-phosphorylated pY(397)pY(407) peptide. These findings will impact significantly on future studies of FAK activity since pY(397) is often used as a measure of FAK activity and Src association. Copyright (c) 2006 John Wiley & Sons, Ltd.