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

Issa, Samah M. A., Schulz, Benjamin L., Packer, Nicolle H. and Karlsson, Niclas G. (2011) Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis. Electrophoresis, 32 24: 3554-3563.


Author Issa, Samah M. A.
Schulz, Benjamin L.
Packer, Nicolle H.
Karlsson, Niclas G.
Title Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis
Journal name Electrophoresis  (ERA 2012 Listed)    (ERA 2010 Rank A)   Check publisher's open access policy
Publication date 2011-12
Sub-type Article
DOI 10.1002/elps.201100374
Volume number 32
Issue number 24
ISSN 0173-0835; 1522-2683
Start page 3554
End page 3563
Total pages 10
Editor Yehia Mechref
Place of publication Weinheim, Germany
Publisher Wiley - VCH Verlag
Collection year 2012
Language eng
Formatted 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.
Keyword Glycomics
Mucin
O-linked glycosylation
Software
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Special Issue: Focus On Glycomics & Glycoproteomics

Document type: Journal Article
Sub-type: Article
Collections: Official 2012 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Thu, 09 Feb 2012, 10:15:35 EST by Lucy O'Brien on behalf of School of Chemistry & Molecular Biosciences