Characterization of the Gla36 residue within the protein S amino acid sequence by mass spectrometry. Protein S was incubated with trypsin, and the subsequent tryptic digest was analyzed by LC-MS/MS using an analytical C18 nanocapillary column plugged to an electrospray quadrupole–time of flight mass spectrometer (ESI Q-STAR). Two distinct species of the EVFENDPETDYFYPK peptide, containing 2 and 3 γ-carboxylated glutamic acid residues, were detected between 33.4 and 33.6 minutes elution time as shown in the TIC chromatogram (A). These 2 peptides were observed in positive ion mode as doubly charged molecular ions with m/z 990.76 and 1012.75, respectively. (One example of the 3 possible combinations of the 2 Gla containing peptide at m/z 990.8 is shown; B.) Both of these molecular ions were subjected to collision-induced fragmentation (parent ion m/z 1012.75 in panel C). The resulting MS/MS spectra show a number of fragment ions, mainly b- and y-type ions, allowing the unambiguous characterization of the peptide amino acid sequence.
