A. Guttman et al., SEPARATION OF 1-AMINOPYRENE-3,6,8-TRISULFONATE-LABELED ASPARAGINE-LINKED FETUIN GLYCANS BY CAPILLARY GEL-ELECTROPHORESIS, Electrophoresis, 17(2), 1996, pp. 412-417
Asparagine-linked glycans of bovine fetuin were separated by capillary
gel electrophoresis after enzymatic release (peptide-N-glycosidase F)
and labeling via reductive amination by a fluorescent dye, 1-aminopyr
ene-3,6-8-trisulfonate (APTS). At low separation pH (2.5) only two dom
inant peaks were observed. Increasing the separation buffer pH to 4.75
resulted in complete separation of two primary doublets and several m
inor peaks from the fetuin N-linked glycan pool. Two of the four major
peaks were spiked with purified individual standards and were identif
ied as trisialylated triantennary structures with different sialylatio
n linkages. The other two larger peaks were postulated to be tetrasial
ylated triantennary structures, based on calculations considering thei
r corresponding glucose unit (GU) values. Effects of the electrophoret
ic separation parameters, such as gel concentration, electric field st
rength and temperature on the migration behavior of the two major doub
lets of the fetuin glycan pool were also thoroughly examined. Our data
suggest that the capillary gel electrophoresis separation of the mult
isialylated branched oligosaccharides with different linkage isomers,
released from bovine fetuin, is fundamentally based on their degree of
sialylation and hydrodynamic volumes.