Study of highly sensitive determination of oligosaccharides by enzymic transglycosylation reaction

采用转糖基化反应和超高效液相色谱-电喷雾飞行时间质谱（UPLC-ESI-TOF-MS）相结合的方法测定糖蛋白中天冬酰胺连接的低聚糖。采用从毛霉中分离的Endo-β- n -乙酰氨基葡萄糖苷酶（Endo- m）进行糖基化酶法测定低聚糖。首次合成了几种荧光天冬酰胺- n-乙酰- d -氨基葡萄糖（Asn-GlcNAc）作为检测糖蛋白中低聚糖的受体。用Endo-M将低聚糖转移到荧光受体上，生成荧光低聚糖。转糖基化活性依赖于Asn-GlcNAc衍生物。其中，从转糖基化率来看，NDA-Asn-GlcNAc是一个合适的受体。NDA-Asn-GlcNAc与Endo-M反应得到的荧光寡糖通过荧光检测的超高效液相色谱（UPLC）从非荧光寡糖中分离得到。采用ESI-TOF-MS对低聚糖进行结构鉴定。溶胶-凝胶技术也成功地实现了Endo-M的固定化。用该方法鉴定了卵清蛋白中的几种低聚糖。采用半微二维(2D)-HPLC-ESI-TOF-MS法测定卵清蛋白中的低聚糖。采用一维酰胺-80色谱柱对荧光标记的低聚糖进行分离。在阴离子交换柱中，荧光低聚糖组分被有效捕获。利用二维ODS色谱柱对捕获的低聚糖进行分离，并用ESI-TOF-MS进行灵敏度测定。用该方法鉴定了从卵清蛋白中分离得到的10个低聚糖。并采用毛细管电泳-TOF-MS法测定低聚糖的含量。结果表明，采用Endo-M和LC-ESI-TOF-MS的方法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，可以实现高通量测定。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