MS/MS spectrum of multiple esterified glutamic acid residues.

(A) Two MS/MS spectra of HC 378–399 with identical m/z showing the esterification at E387 and E389 differentiated by distinct y11+ and y12+ ions. (B) MS/MS spectra of tryptic peptide HC 300–308 showing esterification at either E300 or E301. The highest intensity singly charged ion at m/z 1556.65 is a MS/MS fragment of a peptide containing the N-acetylglucosamine remnant of a liberated N- glycan. Color code: blue square-N-acetylglucosamine; red triangle-fucose; green circle-mannose. (C) MS/MS spectrum of HC 282–295 demonstrates E290 as the site of esterification.</p

General scheme of the esterification reaction of an acid with a sugar alcohol.

General scheme of the esterification reaction of an acid with a sugar alcohol.</p

Confirmation of glutamic acid esterification by sorbitol.

(A) MS spectrum of two species with [M + 3H]+3 ions at approximately equivalent intensity. A m/z difference of 2 was observed in the isotopically labeled sorbitol (13C6) sample. The difference in the fragments’ molecular weight is from the incorporation of the isotopically labeled sorbitol (13C6). (B) MS/MS spectra of tryptic peptide HC 263–281 esterified at E276. The two spectra show the TPEVTCVVVDVSHEDPEVK peptide with the same y and b ions (N-terminal and C-terminal fragments of the peptide are marked as y- and b-ions). The lower spectrum however shows a 6 Da mass difference in the y6+ -y13+ ions; a consequence of esterification from the isotopically labeled sorbitol (13C6) at E276.</p

Esterification levels in each modified peptide.

(A) The esterification percentages of all modified peptides from the 0 week, 1 week, 2 weeks and 4 weeks samples increased over time. (B) The percentage of esterification for all peptides in the 4-week sample with 270mM sorbitol and 270mM 13C6 sorbitol was two to six times higher than in the 4-week sample with 270mM sorbitol alone, representing a two-fold increase in sorbitol concentration.</p

Total Ion Chromatogram (TIC) and MS/MS spectra showing the site- specific identification of esterified glutamic acid residues within the tryptic peptide HC 263–281.

(A) Total Ion Chromatogram showing distinct esterification peaks with identical m/z eluting prior to the unmodified peptide peak. (B) MS/MS spectrum of E279 esterification within the tryptic peptide HC 263–281. Despite sharing an identical parent mass, this peptide possesses a distinct y4+ -y5+ ion series compared to the E276 esterified residue in HC 263–281 (Fig 2B).</p

Summary of observed esterified glutamic acid residues, location and percent esterification.

Summary of observed esterified glutamic acid residues, location and percent esterification.</p