Analyzing glycoproteins separated by two-dimensional gel electrophoresis

Electrophoresis
N H PackerA A Gooley

Abstract

Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosacchari...Continue Reading

References

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Citations

Nov 18, 2000·Electrophoresis·E JungD F Hochstrasser
Sep 18, 1998·Electrophoresis·N H Packer, M J Harrison
Jun 26, 2012·International Archives of Occupational and Environmental Health·Louise FornanderPål Graff
Jan 16, 2007·Journal of Cancer Research and Clinical Oncology·Hai-Jun ZhouPeng-Yuan Yang
Jun 21, 2006·Proteomics·Willy MorelleJean-Claude Michalski
Nov 13, 2008·Journal of Cellular Biochemistry·Jacqueline VazquezMary Ann Greco
Jan 18, 2011·Proteomics·Martin Pabst, Friedrich Altmann
Feb 6, 2004·Journal of Biochemistry and Molecular Biology·Jawon Seo, Kong-Joo Lee
Jan 15, 2016·Electrophoresis·Stefan WeizVéronique Blanchard
Aug 9, 2012·ACS Chemical Biology·Juan EtxebarriaNiels-Christian Reichardt
Apr 12, 2002·Molecular Therapy : the Journal of the American Society of Gene Therapy·Robert PawliukPhilippe Leboulch
Nov 15, 2005·Molecular & Cellular Proteomics : MCP·Geoff W BirrellMartin F Lavin
Sep 25, 2003·Journal of Neuropathology and Experimental Neurology·Ping-Pin ZhengJohan M Kros
Dec 8, 2015·Glycoconjugate Journal·Lucia F Zacchi, Benjamin L Schulz

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