1999 Fiscal Year Final Research Report Summary
Development of methods to determine carbohydrate structure by Fourier transform mass spectrometry
| Project/Area Number |
09558083
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Structural biochemistry
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| Research Institution | Tokyo Metropolitan Institute of Gerontology |
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Principal Investigator |
ENDO Tamao Tokyo Metropolitan Institute of Gerontology, Department of Glycobiology, Head, 糖鎖生物学部門, 研究室長 (30168827)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Tasuku Tokyo Metropolitan Institute of Gerontology, Department of Glycobiology, Researcher, 糖鎖生物学部門, 研究員 (40291132)
SATO Yuji Tokyo Metropolitan Institute of Gerontology, Department of Glycobiology, Researcher, 糖鎖生物学部門, 研究員 (90280768)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Fourier transform mass spectrometry (FTMS) / Glycoprotein / Carbohydrate / post source decay / MALDI-TOM / MS / 2-Aminobenzamide |
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| Research Abstract |
Molecular weight of seven 2-aminobenzamide (2AB)-derivatized oligosaccharides could be determined as small as 10 pmol by matrix-assisted laser desorption ionization (MALDI)-Fourier transform mass spectrometry (FTMS) with a good signal-to-noise ratio. Interestingly, all ions from either high-mannose or complex-type sugar chains have shown spontaneously with the release of GIcNac-3AB by B-type cleavage. Additionally, loss of two hexoses was found from nonreducing terminal from bi-, tri-, and tetra-antennary complex-type sugars, suggesting that galactose residues on GIcNAcβ1-2Man arm may be deleted specifically. On the other hand, ManィイD29ィエD2GIcNAcィイD22ィエD2-2AB released three hexoses from nonreducing termini, suggesting three Manα1-2 linkages may be fragile. 2AB-derivatized oligosaccharides were separated by three lectin column chromatographies and then subjected to MALDI time-of-flight mass spectrometry (MALDI-TOF/MS) for structural characterization of the carbohydrates. The combination
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of sequential exoglyconsidase digestion and MALDI-TOF/MS greatly facilitates the monosaccharide sequencing and is more feasible than size-exclusion column chromatography in terms of the time consumed and the laboriousness of the procedure. By this strategy, microsequencing of 2-3 pmol of oligosaccharide derivatives could be achieved. Furthermore, spectra obtained by the post source decay (PSD) mode provide excellent sequence information. The relative intensities of metastable ions due to fragmentation at glycosidic linkages were different among linkage isomers of particular oligosaccharides. These results demonstrate that PSD analysis possesses significant potential for the estimation of glycosidic linkage in carbohydrate structures. The quantitative nature of MALDI-TOF/MS in oligosaccharide analysis was of great use in the comparative study on normal and pathogenic prion proteins, PrPィイD1cィエD1 and PrPィイD1ScィエD1, respectively. Our developed methods enable analysis of glycan mixtures quantitatively. Less
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