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Analysis of O-linked reducing oligosaccharides released by an in-line flow system

Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/103848

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Title: Analysis of O-linked reducing oligosaccharides released by an in-line flow system
Related: Analytical biochemistry, Vol. 305, Issue 2 (2002), p.173-185
DOI: 10.1006/abio.2002.5657
Publisher: Elsevier
Date: 2002
FoR/RFCD Code(s): 030403 Characterisation of Biological Macromolecules 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics) 030406 Proteins and Peptides
Author/Creator: Karlsson, Niclas G
Author/Creator: Packer, Nicolle H
Description: Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline β-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline β-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS–PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent.
Description: 13 page(s)
Subject Keyword: 030403 Characterisation of Biological Macromolecules
Subject Keyword: 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
Subject Keyword: 030406 Proteins and Peptides
Subject Keyword: oligosaccharides
Subject Keyword: O-linked
Subject Keyword: mucin
Subject Keyword: release
Subject Keyword: fetuin
Subject Keyword: LC-MS
Subject Keyword: graphitized carbon
Resource Type: journal article
Organisation: Macquarie University. Dept. of Chemistry and Biomolecular Sciences

Identifier: http://hdl.handle.net/1959.14/103848
Identifier: ISSN:0003-2697
Identifier: mq-rm-2006010841
Language: eng
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