色谱

色谱

• 样品预处理方法专栏 • 上一篇    下一篇

糖蛋白结构质谱解析的样品前处理

周玮,刘晓慧,周新文,申华莉,杨芃原   

  1. Institutes of Biomedical Sciences, Fudan University, Shanghai 200032,

    China

  • 收稿日期:2007-08-14 修回日期:1900-01-01 出版日期:2007-09-30 发布日期:1985-03-25
  • 通讯作者: 杨芃原

Sample Pre-Processing for Mass Spectrometric Analysis of Glycoprotein

Structure

ZHOU Wei, LIU Xiaohui, ZHOU Xinwen, SHEN Huali, YANG Pengyuan   

  1. Institutes of Biomedical Sciences, Fudan University, Shanghai 200032,

    China

  • Received:2007-08-14 Revised:1900-01-01 Online:2007-09-30 Published:1985-03-25

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摘要：

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(RNase B)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(RNase B适合化学变

性,HRP适合热变性)、酶的选择及条件(RNase B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12～16 h)、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。

关键词: 糖蛋白结构, 条件优化 , 样品前处理, 质谱解析

Abstract:

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, 12-16 h

of digestion, 50% acetonitrile-5% trifluoroacetic acid of the extraction

solution, sandwich spotting method of the sample.

Key words: glycoprotein structure, conditions of optimization , mass spectrometric analysis, sample pre-preparation