Journal Article

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

Fu-Tai A. Chen, Thomas S. Dobashi and Ramon A. Evangelista

in Glycobiology

Published on behalf of Society for Glycobiology

Volume 8, issue 11, pages 1045-1052
Published in print November 1998 | ISSN: 0959-6658
Published online November 1998 | e-ISSN: 1460-2423 | DOI: http://dx.doi.org/10.1093/glycob/8.11.1045
Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

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A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvatelyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 64-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS-monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.

Keywords: 8-aminopyrene-1,3,6-trisulfonic acid (APTS); N-acetylneuraminic acid; monosaccharides; fetuin; capillary electrophoresis with laser-induced fluorescence detection

Journal Article.  3173 words.  Illustrated.

Subjects: Carbohydrates

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