Analytical Chemistry Division, National Institute of Standards and Technology,100 Bureau Drive, Stop 8392, Gaithersburg, MD 20899, USA.
J Mass Spectrom. 2011 Jul;46(7):649-57. doi: 10.1002/jms.1934.
The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.
本项目描述了通过化学酶法修饰牛核糖核酸酶 B（RNase B），使其在一个已知聚糖上含有一个单一的糖基化位点。合成了一种反应性二糖恶唑啉衍生物，并通过内切-β-N-乙酰氨基葡萄糖苷酶 M 催化的化学酶转糖基作用，立体特异性地添加到去糖基化的 RNase B 中。通过质谱优化了恶唑啉的形成条件，并根据其碰撞诱导解离（CID）质谱对产物进行了验证。还使用质谱监测了天然聚糖的酶促去除以及所需均相产物的形成。使用连续四轮 CID 的 LC-MS(n) 用于验证原始糖基化位点已被重组以包含新的聚糖。本文所述的技术不仅限于该分析物或聚糖，并且应该适用于通过明智选择酶/底物组合合成许多具有均一性的糖缀合物。化学酶合成和基于质谱的表征的联合使用为开发均一糖蛋白参考物质展示了前景。含有已知组成、连接和立体化学的单一聚糖的明确糖蛋白标准品对于糖蛋白分析技术的比较和评估将具有重要价值。
