Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion |
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Authors: | W T Wang N C LeDonne B Ackerman C C Sweeley |
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Institution: | 1. Institute of Chemistry, Chinese Academy of Sciences, Zhong Guan Cun, Beijing (Peking), The Peoples Republic of China;2. Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 USA;3. Department of Chemistry, Michigan State University, East Lansing, Michigan 48824 USA |
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Abstract: | A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined. |
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Keywords: | oligosaccharides high-performance liquid chromatography thin-layer chromatography gas-liquid chromatography enzyme digestion fast-atom bombardment-mass spectrometry uv-absorbing derivatives ozonolysis glycosphingolipids |
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