title: Highly sensitive glycosylamine labelling of O-glycans using non-reductive β-elimination.
abstract: When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.
authors.affiliation: Process Lab II, Biotechnology Labs, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2698, Japan.|Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Furuki
authors.affiliation: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
authors.affiliation: Biotechnology Labs, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2698, Japan. kazutoshi.ban@astellas.com.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Ban
mesh_terms: D000588:Amines / Q000032:analysis*; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D057230:Limit of Detection; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000588:Amines; D006020:Glycopeptides; D011134:Polysaccharides
keywords: Bioanalytical methods; Biotechnological products; Fluorescence; HPLC; Mass spectrometry