Quantitative analysis of oligosaccharide structure of glycoproteins |
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Authors: | Kern Hee Chang Tamao Endo Jung Hoe Kim |
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Institution: | (1) Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung dong, Yusung gu, 305-701 Taejon, Korea;(2) Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, 173-0015 Tokyo, Japan |
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Abstract: | A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis
of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate
moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled
with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides
were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment
with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase
HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential
digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major
sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant.
These results coincided well with reported results. |
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Keywords: | hydrazinolysis oligosaccharide 2-aminobenzamide IgG bovine transferrin |
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