Study of highly sensitive determination of oligosaccharides by enzymic transglycosylation reaction

建立了糖蛋白中天冬酰胺低聚糖的转糖基化反应与超高效液相色谱-电喷雾飞行时间质谱仪(UPLC-ESI-TOF-MS)联用的分析方法。低聚糖的测定是基于从毛霉中分离的内切β-N-乙酰氨基葡萄糖苷酶(Endo-M)的酶促糖基化反应。首次合成了几种荧光天冬酰胺-N-乙酰-D-氨基葡萄糖(ASN-GlcNAc)作为测定糖蛋白中寡糖的受体。将寡糖转移到带有Endo-M的荧光受体上，得到荧光低聚糖。ASN-GlcNAc的糖基化活性依赖于其衍生物。其中，NDA-Asn-GlcNAc是一种较好的转糖基化受体。NDA-Asn-GlcNAc与Endo-M反应得到的荧光寡糖为…采用UPLC荧光检测法对非荧光低聚糖进行了检测。用电喷雾飞行时间质谱仪鉴定了低聚糖的结构。采用溶胶凝胶法成功地实现了Endo-M的固定化。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)-高效液相-电喷雾飞行时间-质谱法(ESI-TOF-MS)测定卵清蛋白中寡糖的含量。荧光标记的寡糖通过一维酰胺-80柱分离。荧光低聚糖组分被有效地固定在阴离子交换柱中。捕获的低聚糖用二维ODS柱分离，ESI-TOF-MS灵敏测定。用该方法鉴定了10种从卵清蛋白中分离出来的寡糖。并尝试了毛细管电泳联用飞行时间质谱仪测定低聚糖的含量。基于这一结果，利用Endo-M和LC-ESI-TOF-MS建立的方法可用于寡糖的在线分析。虽然分析运行时间仍然很长，但通过对分离和检测条件的优化，将进行高通量测定。较少

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