A Highly Sensitive Method for Analyses of Sugar Moieties of Glycoproteins by Fluorescence Labeling

Abstract

The sensitivity of a fluorescence labeling method ((1979) J. Biochem. 85, 989–994; 995–1002) for structure analyses of asparagine-linked sugar moieties of glycoproteins was increased by using HPLC with a fluorescence detector. Sugar moieties were separated from polypeptide portions by hydrazinolysis. Free amino groups thus exposed were acetylated and the reducing ends of sugar chains were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the use of sodium cyano-borohydride. The pyridylamino derivatives were purified on a Dowex 1 column to eliminate undesired substances. The separation and identification of the pyridylamino derivatives were carried out by HPLC with a column of C18 reversed phase or gel permeation phase. As little as 0.1 pmol of pyridylamino derivatives can be detected. Ten microgram of Taka-amylase A was easily detected by this system. The method was also applied to some other glycoproteins.