Proteome analysis of glycoforms: A review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Nicolle H. Packer*, Anna Pawlak, Warren C. Kett, Andrew A. Gooley, John W. Redmond, Keith L. Williams

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Citations (Scopus)

Abstract

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from posttranslational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels.

Original languageEnglish
Pages (from-to)452-460
Number of pages9
JournalElectrophoresis
Volume18
Issue number3-4
DOIs
Publication statusPublished - Mar 1997

Keywords

  • Gel electrophoresis
  • Glycoprotein
  • Oligosaccharides
  • Post-translational modifications
  • Proteome
  • Two-dimensional polpacrylamide

Fingerprint

Dive into the research topics of 'Proteome analysis of glycoforms: A review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis'. Together they form a unique fingerprint.

Cite this