Sensitive analysis of O-linked sugar chains by fluorescence labeling

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Sugar chains linked to serine or threonine residues of glycoproteins through and O-glycosidic bond are found in mucin, immunoglobulin, hormonos, etc. To liberate O-linked sugar chains, a method based on alkaline-borohydride treatment is mostly used. We have developed a fluorescence labeling method for highly sensitive detection of sugar chains and applied the method to analyze N-linked sugar chains. In this study we applied the method to Oーlinked sugar chains.Sugar chains liberated by alkaline-borohydride have no reducing end aldehyde group, therefore fluorescence labeling of the sugar chains is not possible. So a new method is required to tag the reducing end of sugar chains with 2-aminopyridine. Fot this purpose we used hydrazinolysis. To analyze the liberation of sugar chains quantitatively, we prepared glycopeptide having two O-linked sugar chains and fluorescence labeled sugar chains. These structures were identified by gas chromatography-mass spectrometry. Sugar chains were liberated by hydrazinolysis of the glycopeptide and sugar chains thus obtained were analyzed as their pyridylamino derivatives by HPLC.Under the established conditions, sugar chains were liberated from glycopeptides in a yield of 80%. The method was applied to an glycoprotein and pyridylamino derivatives of sugar chains were detected by HPLC.