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

Biochimica et biophysica acta (2011-08-02)
Gerhild Zauner, Carolien A M Koeleman, André M Deelder, Manfred Wuhrer
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethylamine hydrochloride, 99%
Sigma-Aldrich
Dimethylamine solution, 40 wt. % in H2O
Sigma-Aldrich
Dimethylamine, anhydrous, ≥99%
Sigma-Aldrich
Dimethylamine solution, 2.0 M in THF
Sigma-Aldrich
Dimethylamine solution, 2.0 M in methanol
Sigma-Aldrich
Dimethylamine hydrochloride, purum, ≥98.0% (AT)