ABSTRACT
Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (â¼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.
