糖蛋白结构质谱解析的样品前处理

Sample Pre-Processing for Mass Spectrometric Analysis of Glycoprotein Structure

基于高分辨率、高精度、高灵敏度及良好表现力的多级质谱,以标准糖蛋白辣根过氧化物酶(HRP)、核糖核酸酶B(RNaseB)为对象,探索了糖蛋白质谱分析前样品处理的技术流程。通过对糖蛋白样品前处理的各个步骤(包括蛋白变性方法(RNaseB适合化学变性,HRP适合热变性)、酶的选择及条件(RNaseB适用内蛋白酶Lys-C酶解,HRP适用胰蛋白酶酶解,酶解时间控制在12～16h)、提肽液的组成(对于低丰度糖蛋白用50%乙腈-5%三氟乙酸提取)、基质的组成、点样方法("三明治"式))进行了优化,形成了简单易行、无需糖肽富集标记的糖蛋白溶液酶解-质谱分析和胶上分离酶解-质谱分析的技术路线,为实际样品体系中糖蛋白的结构解析提供了可行的方法。

A novel, simple and fast method based on a quadrupole ion trap （QIT） and a time- of-flight mass spectrograph was developed for the glycosylation site and glycan structure analysis of glycoprotein by in-solution digestion and in-gel digestion which did not need to enrich and label the glycopeptide. Horseradish peroxidase （HRP） and RNase B were utilised to optimize the conditions of sample pre-processing. The effects of the conditions were investigated separately and the optimal conditions were ： heat denaturation of RNase B, chemical denaturation of HRP, RNase B digestion with endoproteinase Lys-C, HRP digestion with trypsin, 12 - 15 h of digestion, 50% acetonitrile-5% trifluoroacetic acid of the extraction solution, sandwich spotting method of the sample.