- 作者：Gerhild Zauner ; Carolien A.M. Koeleman ; Andr¨¦ M. Deelder ; Manfred Wuhrer
- 关键词：Glycoproteins ; Mass spectrometry ; O-glycans ; Oligosaccharides
- 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
- 出版年：2012
- 出版时间：September, 2012
- 年：2012
- 卷：1820
- 期：9
- 页码：1420-1428
- 全文大小：1083 K
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.
General Significance
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.