Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharidesand glycoproteins is presented. The analysis is based on the combination ofchemical and enzymatic methods coupled with capillary electrophoretic (CE)separation and laser-induced fluorescence (LIF) detection. The presentmethod utilizes a simplified acid hydrolysis procedure consisting of mildhydrolysis (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 fromstrong acid hydrolysis of oligosaccharides and glycoproteins werereacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS)along with neutral sugars in the presence of sodium cyanoborohydride toyield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalianglycoproteins, was converted in a fast specific reaction by the action ofneuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) toN-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized withAPTS in the same manner as the other monosaccharides. This method wasdemonstrated for the quantitation of pure Neu5Ac and the species derivedfrom mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovinefetuin glycan. Quantitative recovery of the N-acetylmannosamine wasobtained from a known amount of Neu5Ac in a mixture of seven othermonosaccharides or from the sialylated oligosaccharides occurring inglycoproteins. The sequence of procedures consists of acid hydrolysis,enzymatic conversion and APTS derivatization which produced quantitativerecovery of APTS- monosaccharide adducts. The detection limits for sugarsderivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50pmol for the other sugars.