Study of highly sensitive determination of oligosaccharides by enzymic transglycosylation reaction

酶转糖基反应高灵敏测定低聚糖的研究

• 批准号: 17590037
• 负责人: TOYO'OKA Toshimasa
• 金额: $ 2.24万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590037/
• 关键词: 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

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 … More 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

采用转糖基反应和超高效液相色谱-电喷雾电离飞行时间质谱（UPLC-ESI-TOF-MS）相结合的方法测定糖蛋白中的天冬酰胺连接寡糖。寡糖的测定基于与从冬毛霉中分离的内切-β-N-乙酰氨基葡萄糖苷酶 (Endo-M) 的酶转糖基反应。首先合成了几种荧光天冬酰胺酰-N-乙酰基-D-葡萄糖胺（Asn-GlcNAc）作为受体用于测定糖蛋白中的寡糖。用Endo-M将寡糖转移到荧光受体上以产生荧光寡糖。转糖基活性取决于 Asn-GlcNAc 衍生物。其中，就转糖基化产率而言，NDA-Asn-GlcNAc是合适的受体。 NDA-Asn-GlcNAc 与 Endo-M 反应获得的荧光寡糖是通过 UPLC 和荧光检测从非荧光寡糖中分离出来的。然后通过 ESI-TOF-MS 鉴定寡糖的结构。 Endo-M 的固定化也通过溶胶-凝胶技术成功进行。通过所提出的方法鉴定了卵清蛋白中的几种寡糖。卵清蛋白中寡糖的测定也通过半微量二维 (2D)-HPLC-ESI-TOF-MS 进行。荧光标记的寡糖通过一维Amide-80柱分离。荧光寡糖的部分被有效地捕获在阴离子交换柱中。然后通过二维 ODS 柱分离捕获的寡糖，并通过 ESI-TOF-MS 灵敏地测定。从所提出的方法中鉴定出从卵清蛋白中释放出的十种寡糖。还尝试通过毛细管电泳 (CE)-TOF-MS 测定寡糖。根据结果​​，所提出的利用 Endo-M 和 LC-ESI-TOF-MS 的方法可能适用于在线寡糖分析。尽管分析运行时间仍然较长，但通过优化分离和检测条件将进行高通量测定。较少的