SEPARATION OF 1-AMINOPYRENE-3,6,8-TRISULFONATE-LABELED ASPARAGINE-LINKED FETUIN GLYCANS BY CAPILLARY GEL-ELECTROPHORESIS

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.