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Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis

Journal article
Authors SMA Issa
BL Schulz
NH Packer
Niclas G. Karlsson
Published in ELECTROPHORESIS
Volume 32
Issue 24
Pages 3554-3563
ISSN 0173-0835
Publication year 2011
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 3554-3563
Language en
Links dx.doi.org/10.1002/elps.201100374
Keywords glycomics, mucin, o-linked glycosylation, software
Subject categories Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Abstract

Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.

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