A highly sensitive method for analyses of sugar moieties of glycoproteins by fluorescence labeling.
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 cyanoborohydride. 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.
D000631:Aminopyridines; D001894:Borohydrides; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003486:Cyanides; D004187:Disaccharides; D005456:Fluorescent Dyes; D005936:Glucans / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006834:Hydrazines; D009844:Oligosaccharides / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000032:analysis
D000631:Aminopyridines; D001894:Borohydrides; D003486:Cyanides; D004187:Disaccharides; D005456:Fluorescent Dyes; D005936:Glucans; D006023:Glycoproteins; D006834:Hydrazines; D009844:Oligosaccharides; C029424:hydrazine; C032438:chitobiose; C009282:sodium cyanoborohydride; D000516:alpha-Amylases; C032439:alpha-aminopyridine