HIGH-RESOLUTION CAPILLARY GEL-ELECTROPHORESIS OF REDUCING OLIGOSACCHARIDES LABELED WITH 1-AMINOPYRENE-3,6,8-TRISULFONATE

High-resolution capillary gel electrophoresis was used for the separat ion of oligosaccharides labeled with a novel fluorophore 1-aminopyrene -3,6,8-trisulfonate (APTS) at the reducing termini by reductive aminat ion. The APTS-saccharide adducts were detected by laser-induced fluore scence with excitation by the 488-nm Ar-ion laser and a 520-nm emissio n filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Deriv atization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent ar e thoroughly discussed. Desialylation of several sialylated oligosacch arides with different structures and linkages due to the effects of la beling temperature and time are also addressed. Employing the optimize d conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separate d and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrop horesis separation of N-linked glycans of ribonuclease-B and fetuin ar e also shown. (C) 1996 Academic Press, Inc.