通用的糖组内标制备方法和精准糖组定量研究

糖链标志物和功能研究及生物药质控等领域亟需实用的糖组定量方法。质谱是分析复杂糖组最有效手段之一。常以内标校正质谱分析，但简单的内标组成难以实现种类和丰度多样的糖组的准确校正，申请人曾在匀相中把N-糖的半缩醛基一步还原成氘代醇羟基，得到与实际N-糖组糖链数目、种类及丰度均相似的糖组内标，实现了N-糖组精准定量，但该方法不适于O-糖组定量。本项目拟通过氨基反应把糖蛋白固定在醛基固体上再进行唾液酸保护，然后用糖苷酶释放N-糖组并一步还原成氘代醇羟基制备N-糖组内标；再非还原性β-消除释放制备半缩醛羟基O-糖组内标。采用相似的简单流程依次获得样品中的半缩醛基N-糖组和氘代醇羟基O-糖组，针对O-糖组仅改用还原性β-消除结合NaBD4。本项目仅通过成熟的反应在同一流程中可为糖组中每个糖链提供量身定做的内标，实现N-糖组和O-糖组简便、通量、准确的定量，为挖掘胰腺癌等疾病糖链标志物提供有效方法和数据。

Practical glycome quantitative methods are urgently needed in the fields of disease marker discovery, protein glycosylation function research and quality control of biopharmaceuticals. Mass spectrometry is one of the most effective methods for protein glycosylation analysis. Internal standard is commonly used to reduce the errors induced by fluctuation in the ionization efficiencies of various glycans and instrument response among different runs. However, simple kinds of internal standard cannot accurately correct complex glycans in glycome. The applicant has reduced the hemiacetal group of N-glycans to deuterated alditol group in liquid homogenization phase and synthesized the internal standard of N-glycome which was similar in glycan number, composition and abundance to the N-glycome to be analyzed, finally achieving precise N-glycome quantitation. However, this method is not applicable for O-glycome quantitation. In this project, the glycoprotein was immobilized on an aldehyde solid by amide reaction followed by sialic acid protection, and then the N-glycome was released by glycosidase and reduced to deuterated alditol group in one step to prepare the N-glycome internal standard. The internal standard of O-glycome was prepared with non-reducing elimination which has the hemiacetal hydroxyl group in the reducing end. A similar simple process was used to obtain the hemiacetal N-glycome and deuterated alditol O-glycome in the sample in turn. Only the reductive β-elimination combined with NaBD4 was used for the O-glycome. This project provides a general method for preparation of internal standard for each glycan in the glycome in one pipeline only through mature reactions and hence developed a simple, high-throughput and accurate quantification of N- glycome and O-glycome, which provides an effective method to discover glycan markers of pancreatic cancer and other diseases.