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.