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 separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS-saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized 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 separated and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown.
Guttman
A
A
Beckman Instruments, Inc., Fullerton, California 92634, USA.
Chen
F T
FT
Evangelista
R A
RA
Cooke
N
N
D000117:Acetylglucosamine / Q000737:chemistry; D002240:Carbohydrate Sequence; D002250:Carbon Radioisotopes; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction; D011721:Pyrenes / Q000737:chemistry*
C098210:1-aminopyrene-3,6,8-trisulfonic acid; D002250:Carbon Radioisotopes; D009844:Oligosaccharides; D011721:Pyrenes; D000117:Acetylglucosamine