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Analyzing glycoproteins separated by two-dimensional gel electrophoresis
N H Packer1, M A Lawson, D R Jardine
1Macquarie University Center for Analytical Biotechnology, School of Biological Sciences and Chemistry, Macquarie University, Sydney, NSW, Australia. nicolle.packer@mq.edu.au
Electrophoresis
|June 25, 1998
Summary
Analyzing glycoprotein spots using 2-D electrophoresis reveals glycosylation patterns. Understanding these patterns helps predict changes in protein glycosylation, crucial for glycoprotein analysis.
Area of Science:
- Proteomics
- Glycomics
- Biochemistry
Background:
- Two-dimensional (2-D) electrophoresis is key for separating protein glycoforms.
- Glycoprotein isoforms often appear as 'trains' of spots and can vary in molecular weight.
- Understanding migration rules in 2-D electrophoresis is vital for interpreting glycosylation patterns.
Purpose of the Study:
- To describe techniques for analyzing oligosaccharides from glycoproteins separated by 1-D and 2-D electrophoresis.
- To establish rules governing glycoprotein migration in 2-D electrophoresis for predictive analysis.
- To interpret changes in glycosylation patterns by analyzing carbohydrate content.
Main Methods:
- Enzymatic (PNGase F) or chemical (beta-elimination) release of oligosaccharides from PVDF membranes.
- Separation of oligosaccharides by high-performance anion-exchange chromatography (HPAEC-PAD).
- Identification via electrospray ionization mass spectrometry (ESI-MS) and protein analysis by Edman sequencing or acid hydrolysis.
Main Results:
- Demonstrated techniques on bovine fetuin and human glycophorin A.
- Applied methods to analyze alpha2-HS glycoprotein and alpha1-antitrypsin in human plasma.
- Confirmed that both macroheterogeneity and microheterogeneity of glycosylation influence 2-D PAGE separation.
Conclusions:
- Developed and validated a comprehensive method for glycoprotein glycoform analysis.
- Provided insights into the contribution of glycosylation heterogeneity to protein separation in 2-D electrophoresis.
- Established a foundation for predictive modeling of glycoprotein behavior in 2-D PAGE.