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Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/103909
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- Title
- Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins
- Related
- Journal of proteome research, Vol. 1, No. 6 (2002), p.521-529
- DOI
- 10.1021/pr025538d
- Publisher
- American Chemical Society
- Date
- 2002
- Author/Creator
- Wilson, Nicole L
- Author/Creator
- Schulz, Benjamin L
- Author/Creator
- Karlsson, Niclas G
- Author/Creator
- Packer, Nicolle H
- Description
- A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive β-elimination and analysis using LC−ESI−MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, α₂-HS-glycoprotein, serotransferrin, α₁-antitrypsin, and α₁-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.
- Description
- 9 page(s)
- Subject Keyword
- 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
- Subject Keyword
- 030403 Characterisation of Biological Macromolecules
- Subject Keyword
- 030406 Proteins and Peptides
- Subject Keyword
- sialylation
- Subject Keyword
- glycoproteins
- Subject Keyword
- β-elimination
- Subject Keyword
- PNGase F
- Subject Keyword
- plasma
- Resource Type
- journal article
- Organisation
- Macquarie University. Department of Chemistry and Biomolecular Sciences
- Identifier
- http://hdl.handle.net/1959.14/103909
- Identifier
- mq:10970
- Identifier
- ISSN:1535-3893
- Identifier
- mq-rm-2006010837
- Language
- eng
- Reviewed
Macquarie University ResearchOnline
