title: Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping.
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 (∼0.1 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.
authors: Department of Biologics Process and Product Development, Bristol-Myers Squibb Company, 6000 Thompson Road, East Syracuse, NY 13057, USA. jacob.bongers@bms.com
authors: Jacob
authors: J
authors: Bongers
authors: John
authors: J
authors: Devincentis
authors: Jinmei
authors: J
authors: Fu
authors: Peiqing
authors: P
authors: Huang
authors: David H
authors: DH
authors: Kirkley
authors: Kirk
authors: K
authors: Leister
authors: Peiran
authors: P
authors: Liu
authors: Richard
authors: R
authors: Ludwig
authors: Kathleen
authors: K
authors: Rumney
authors: Li
authors: L
authors: Tao
authors: Wei
authors: W
authors: Wu
authors: Reb J
authors: RJ
authors: Russell
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D060908:CTLA-4 Antigen / Q000737:chemistry* / Q000378:metabolism; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D006020:Glycopeptides; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014357:Trypsin