Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection.
The method of anion exchange chromatography followed by pulsed amperometric detection (AE-PAD; Johnson, D. C., and Polta, T. Z. (1986) Chromatogr. Forum 1, 37-44) has been applied to the compositional analysis of glycoconjugates. Using 22 mM NaOH as a column effluent, underivatized fucose, galactosamine, glucosamine, galactose, glucose, and mannose were readily separated in 15 min at a flow rate of 1 ml/min. The limit of quantification of the monosaccharides was better than 100 pmol (signal to noise ratio 184:1). AE-PAD was employed to quantify the monosaccharides of several glycoproteins, glycopeptides, and oligosaccharides after hydrolysis with 2 M trifluoroacetic acid. Both neutral and amino sugars could be rapidly estimated in a single chromatographic step using AE-PAD. Complete release of N-acetylglucosamine required more vigorous hydrolysis conditions (Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, pp. 63-73, Academic Press, New York). In both glycopeptides and oligosaccharides, approximately one less residue of Man than predicted was determined. Both AE-PAD and liquid chromatographic analysis of borate-monosaccharide complexes with fluorometric detection (Mikami, H., and Ishida, Y. (1983) Bunseki Kagaku 32, E207-E210) gave similar quantification of mannose and other sugars. The capability of rapid, sensitive quantification of underivitized monosaccharides should facilitate structural analysis of glycoconjugates.
Hardy
M R
MR
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Townsend
R R
RR
Lee
Y C
YC
D000606:Amino Sugars / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000032:analysis*; D006868:Hydrolysis; D009005:Monosaccharides / Q000032:analysis*
D000606:Amino Sugars; D002241:Carbohydrates; D006023:Glycoproteins; D009005:Monosaccharides