Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyl-5-pyrazolone labeling

• Published in: Biochimica et biophysica acta Vol. 1820; no. 9; pp. 1420 - 1428
• Main Authors: Zauner, Gerhild, Koeleman, Carolien A.M., Deelder, André M., Wuhrer, Manfred
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2012
• Subjects: Animals; Antipyrine - analogs & derivatives; Antipyrine - chemistry; Antipyrine - pharmacology; bile salts; breast milk; Carbohydrate Sequence; Cattle; Chromatography, Liquid; dimethylamine; Dimethylamines - pharmacology; Glycomics - methods; Glycoproteins; glycosylation; Humans; Hydrolysis; Mass spectrometry; Mass Spectrometry - methods; Models, Biological; Molecular Sequence Data; mucins; Mucins - chemistry; Mucins - metabolism; O-glycans; Oligosaccharides; polysaccharides; Polysaccharides - analysis; Polysaccharides - chemistry; Polysaccharides - metabolism; Staining and Labeling - methods; stoichiometry; Submandibular Gland - chemistry; Submandibular Gland - metabolism; Glycoproteins; Oligosaccharides; Mass spectrometry; O-glycans
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2011.07.004

Analysis of protein glycosylation is an important first step towards establishing the functions of glycans in health and disease. In contrast to N-glycans which are generally enzymatically released for analysis, there is no corresponding enzyme for O-glycan liberation. Therefore, O-glycans are generally released by chemical methods involving tedious procedures. Here, a straightforward method for the combined release and labeling of O-linked glycans from glycoproteins is described. Dimethylamine serves as the releasing agent, and 1-phenyl-3-methyl-5-pyrazolone (PMP) is employed for a prompt reaction with the reducing end of the freshly released O-glycan structures via an aldol condensation followed by a Michael-type addition resulting in a 2:1 stoichiometry of PMP per glycan. Samples are analyzed by nanoLC coupled to mass spectrometry. Mucin from bovine submaxillary gland was used as a model protein to evaluate and optimize the approach that was further applied to bile salt stimulated lipase (BSSL) isolated from human milk. Next to previously reported O-glycan structures two additional oligosaccharides could be detected for BSSL. In conclusion, the facile protocol established is suitable for the analysis of complex O-linked oligosaccharides from various biological samples. This article is part of a Special Issue entitled Glycoproteomics. [Display omitted] ► O-glycan release by beta-elimination is combined with reducing end labeling. ► A fast method of O-glycan analysis using LC–MS detection is established. ► O-glycan isomer separation and characterization are achieved by reverse phase LC–MS.