title: LC-MS/MS peptide mapping with automated data processing for routine profiling of N-glycans in immunoglobulins.
abstract: Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
authors.affiliation: Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320, USA.
authors.forename: Bhavana
authors.initials: B
authors.lastname: Shah
authors.forename: Xinzhao Grace
authors.initials: XG
authors.lastname: Jiang
authors.forename: Louise
authors.initials: L
authors.lastname: Chen
authors.forename: Zhongqi
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006020:Glycopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D010446:Peptide Fragments; D011134:Polysaccharides