PROTEOME ANALYSIS OF GLYCOFORMS - A REVIEW OF STRATEGIES FOR THE MICROCHARACTERISATION OF GLYCOPROTEINS SEPARATED BY 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isof orms to be readily purified for subsequent analysis, The profile of th e 2-D separation of the protein complement (proteome) of eukaryotic ce lls and tissues typically contains obvious 'trains' of spots which dif fer in pi and/or apparent molecular mass. These are usually isoforms o f the same protein and result from posttranslational modifications. Th ere is growing evidence that alterations to the glycosylation and/or p hosphorylation of a protein call be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separ ation, II is not clear, however, bow these modifications alter the str uctural properties of the protein and affect their migration in this m ode of separation. Strategies need to be developed to obtain a more de tailed understanding of the reason for the appearance of isoforms as d iscrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, we re 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 modi fication. To further investigate the reasons for the different migrati on of the isoforms it is necessary to characterise the modifications i n more detail. Unlike protein analysis, until recently the available m ethodology for the analysis of the glycans attached to proteins has no t been sensitive enough to allow analysis of single spots in gels or b lots resulting from 2-D electrophoresis. In this paper we review curre nt and future strategies for characterisation of protein modifications using single spots from 2-D gels.