QUANTITATIVE-ANALYSIS OF SUGAR CONSTITUENTS OF GLYCOPROTEINS BY CAPILLARY-ELECTROPHORESIS

A method for quantitative analysis of monosaccharides including N-acet ylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combinat ion of chemical and enzymatic methods coupled with capillary electroph oretic (CE) separation and laser-induced fluorescence (LIE) detection. The present method utilizes a simplified acid hydrolysis procedure co nsisting of mild hydrolysis (0.1 M TFA) to release sialic acid and str ong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. A mino sugars released from strong acid hydrolysis of oligosaccharides a nd 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 flu orescent APTS adducts, N-acetylneuraminic acid (Neu5Ac), a major compo nent 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 pyruvat e. ManNAc was then derivatized with APTS in the same manner as the oth er monosaccharides, This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6' -sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative rec overy of the N-acetylmannosamine was obtained from a known amount of N eu5Ac in a mixture of seven other monosaccharides or from the sialylat ed oligosaccharides occurring in glycoproteins, The sequence of proced ures consists of acid hydrolysis, enzymatic conversion and APTS deriva tization which produced quantitative recovery of APTS-monosaccharide a dducts, The detection Limits for sugars derivatized with APTS and dete cted by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugar s.