title: Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyl-5-pyrazolone labeling.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Postbus 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: Gerhild
authors.initials: G
authors.lastname: Zauner
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000818:Animals; D000983:Antipyrine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002417:Cattle; D002853:Chromatography, Liquid; D004123:Dimethylamines / Q000494:pharmacology; D000077553:Edaravone; D054794:Glycomics / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013363:Submandibular Gland / Q000737:chemistry / Q000378:metabolism
chemical_list: D004123:Dimethylamines; D009077:Mucins; D011134:Polysaccharides; C034516:dimethylamine; D000077553:Edaravone; D000983:Antipyrine