PROTEOME ANALYSIS OF GLYCOFORMS - A REVIEW OF STRATEGIES FOR THE MICROCHARACTERISATION OF GLYCOPROTEINS SEPARATED BY 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS
Nh. Packer et al., PROTEOME ANALYSIS OF GLYCOFORMS - A REVIEW OF STRATEGIES FOR THE MICROCHARACTERISATION OF GLYCOPROTEINS SEPARATED BY 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS, Electrophoresis, 18(3-4), 1997, pp. 452-460
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.