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. 2008 Dec 19;75(3):658–666. doi: 10.1124/mol.108.052464

Fig. 4.

Fig. 4.

LC-MRM/MS analysis of activation loop and control peptides from digested endogenous and [13C15N]FAK immunoprecipitated from pervanadate-treated MEFs expressing normal Src and their corresponding heavy isotope-labeled internal standards. Shown are ion chromatograms for peptides resulting from the trypsin hydrolysis of immunoprecipitated endogenous (∼) and [13C15N]FAK (**), spiked with [13C15N]labeled peptide standards (*). MRM transitions monitored the unmodified activation loop peptide (YMEDSTY576Y577K, tR = 16.49 min) in the unlabeled (m/z 600.25 → 905.40), 13C33 15N2-labeled (m/z 617.25 → 930.40), and 13C6 15N2-labeled (m/z 604.25 → 913.40) species. Mono-phosphorylated (pY576) peptides (tR = 15.58 min) were monitored by transitions for the unlabeled (m/z 640.25 → 985.35 and 967.35), 13C33 15N2-labeled (m/z 657.25 → 1010.36 and 992.36), and 13C6 15N2-labeled (m/z 644.25 → 993.36 and 975.36) species. Bis-phosphorylated (pTyr576/pTyr577) peptides (tR = 14.11) were monitored by transitions for the unlabeled (m/z 680.25 → 1047.31), 13C33 15N2-labeled (m/z 697.25 → 1072.32), and 13C6 15N2-labeled (m/z 684.25 → 1055.32) species. Finally, the control segment peptides (E956VGLALR962, tR = 18.62) were monitored by transitions for the unlabeled (m/z 379.23 → 359.24), 13C18 15N6-labeled (m/z 382.23 → 359.24), and 13C5 15N-labeled (m/z 391.23 → 376.24) species.