Study of highly sensitive determination of oligosaccharides by enzymic transglycosylation reaction

将转糖基化反应与超高效液相色谱-电喷雾飞行时间质谱（UPLC-ESI-TOF-MS）相结合，对糖蛋白中天冬酰胺连接寡糖进行了测定。寡糖的测定是基于与从冬季毛霉中分离的内切-β-N-乙酰氨基葡萄糖苷酶（Endo-M）的酶促转糖基反应。首次合成了几种荧光受体天冬酰胺-N-乙酰-D-氨基葡萄糖（Asn-GlcNAc），用于糖蛋白中寡糖的测定。用Endo-M将寡糖转移到荧光受体上以产生荧光寡糖。转糖基化活性依赖于Asn-GlcNAc衍生物。其中，NDA-Asn-GlcNAc是一个合适的转糖基化受体。从NDA-Asn-GlcNAc与Endo-M反应获得的荧光寡糖特异性地被 关于我们 通过具有荧光检测的UPLC从非荧光寡糖分离。采用溶胶-凝胶法成功地固定化了Endo-M。用该方法对卵清蛋白中的几种寡糖进行了鉴定。采用半微量二维（2D）-HPLC-ESI-TOF-MS对卵清蛋白中的寡糖进行检测，荧光标记的寡糖经Amide-80柱分离。阴离子交换柱能有效地捕获荧光寡糖组分。捕获的寡糖，然后通过二维ODS柱分离和ESI-TOF-MS的灵敏度测定。从卵清蛋白释放的寡糖，从所提出的方法鉴定了10个。本文还尝试了毛细管电泳（CE）-飞行时间质谱（TOF-MS）联用技术测定低聚糖，结果表明，Endo-M和LC-ESI-TOF-MS联用技术可用于低聚糖的在线分析。虽然分析运行时间仍然很长，但通过优化分离和检测条件将进行高通量测定。少

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