title: Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry.
abstract: A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.
authors.affiliation: Waters Corporation, Milford, MA 01757, USA. Martin_Gilar@waters.com
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.forename: Ying-Qing
authors.initials: YQ
authors.lastname: Yu
authors.forename: Joomi
authors.initials: J
authors.lastname: Ahn
authors.forename: Hongwei
authors.initials: H
authors.lastname: Xie
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Han
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002240:Carbohydrate Sequence; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays; D000509:alpha-Fetoproteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010455:Peptides; D011134:Polysaccharides; D000509:alpha-Fetoproteins; D014357:Trypsin