Study of highly sensitive determination of oligosaccharides by enzymic transglycosylation reaction
| Project/Area Number |
17590037
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | University of Shizuoka |
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Principal Investigator |
TOYO'OKA Toshimasa University of Shizuoka, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (40183496)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Oligosaccharide analysis / Transglycosylation reaction / Endo-M / Fluorescence acceptor / Ovalbumin / Liquid chromatography / Two dimensional HPLC / ESI-TOF-MS / ESI-TOF-MS / 糖転移酵素 / TOF-MS / キャピラリー電気泳道 |
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| Research Abstract |
The determination of asparagine-linked oligosaccharides in glycoproteins was carried out by combination of the transglycosylation reaction and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The determination of oligosaccharides is based on the enzymic transglycosylation reaction with Endo-β-N-acetylglucosaminidase (Endo-M) isolated from Mucor hiemalis. Several fluorescent asparaginyl-N-acetyl-D-glucosamines (Asn-GlcNAc) were first synthesized as the acceptors for the determination of oligosaccharides in glycoproteins. The oligosaccharides were transferred to the fluorescent acceptors with Endo-M to produce the fluorescent-oligosaccharides. The transglycosylation activity was dependent upon the Asn-GlcNAc derivatives. Among them, NDA-Asn-GlcNAc was a suitable acceptor in terms of transglycosylation yield. The fluorescent oligosaccharides obtained from the reaction of NDA-Asn-GlcNAc with Endo-M were specifically is
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olated from the non-fluorescent oligosaccharides by UPLC with fluorescence detection. The structures of oligosaccharides were then identified by ESI-TOF-MS. The immobilization of Endo-M was also successfully performed by sol-gel technique. Several oligosaccharides in ovalbumin were identified by the proposed method. The determination of oligosaccharides in ovalbumin was also performed by semi-micro two-dimensional (2D)-HPLC-ESI-TOF-MS. The fluorescence labeled oligosaccharides were separation by 1st dimension Amide-80 column. The fraction of fluorescent oligosaccharides was effectively trapped in anion exchange column. The trapped oligosaccharides were then separated by 2nd dimension ODS column and sensitively determined by ESI-TOF-MS. Ten oligosaccharides liberated from ovalbumin were identified from the proposed method. The determination of oligosaccharides was also tried by capillary electrophoresis (CE)-TOF-MS. Based on the results, the proposed method utilizing Endo-M and LC-ESI-TOF-MS may be useful for on-line oligosaccharide analyses. Although the analytical run time is still long, a high-throughput determination will be performed by optimization of the separation and detection conditions. Less
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Research Products
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