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Glycobiology Advance Access originally published online on March 25, 2009
Glycobiology 2009 19(7):756-766; doi:10.1093/glycob/cwp048
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Jessica M Holmén Larsson, Hasse Karlsson, Henrik Sjövall and Gunnar C Hansson1

Departments of Medical Biochemistry and Medicine, University of Gothenburg, Gothenburg, Sweden

1 To whom correspondence should be addressed: Tel: +46-31-7863488; Fax: +46-31-416108; e-mail: gunnar.hansson{at}medkem.gu.se

Received on May 14, 2008; revised on March 16, 2009; accepted on March 20, 2009

High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (~5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS–agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive β-elimination and analyzed by nanoLC/mass spectrometry and MSn. The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Galβ1-3/4-GlcNAcβ1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were Formula -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

Key words: colon / glycan / large intestine / mass spectrometry / oligosaccharide


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