色谱

色谱 ›› 2013, Vol. 31 ›› Issue (11): 1057-1063.DOI: 10.3724/SP.J.1123.2013.05043

• 研究论文 • 上一篇    下一篇

利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型

马成1,2, 潘一廷1,2, 张琪3, 王继峰2, 钱小红1,2, 应万涛2   

  1. 1. 北京理工大学生命学院, 北京 100081;
    2. 蛋白质组学国家重点实验室, 北京蛋白质组研究中心, 军事医学科学院放射与辐射医学研究所, 北京 102206;
    3. 新加坡南洋理工大学生命学院, 新加坡 637551
  • 收稿日期:2013-05-27 修回日期:2013-08-26 出版日期:2013-11-28 发布日期:2013-12-05
  • 通讯作者: 应万涛
  • 基金资助:

    国家重点基础研究"973"项目(2011CB910603);国家高技术研究发展计划项目(2012AA020203);北京市科技新星计划项目(Z121107002512014).

Analysis of N-glycosylated protein and N-glycans in human plasma by hydrophilic enrichment strategy

MA Cheng1,2, PAN Yiting1,2, ZHANG Qi3, WANG Jifeng2, QIAN Xiaohong1,2, YING Wantao2   

  1. 1. School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, China;
    2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China;
    3. School of Biological Sciences, Nanyang Technological University, 637551, Singapore
  • Received:2013-05-27 Revised:2013-08-26 Online:2013-11-28 Published:2013-12-05

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摘要：

蛋白质的N-糖基化是最重要的翻译后修饰之一,许多已知的血浆肿瘤诊断标志物及治疗靶标都是N-糖基化蛋白。针对血浆的糖蛋白质组研究有利于发现新的蛋白标志物。然而,血浆蛋白质浓度分布的动态范围非常宽,且同一位点上的糖链存在微观不均一性,影响了血浆中糖蛋白的鉴定效率。本文利用亲水材料ZIC-HILIC制备亲水富集柱分别对人血浆中的N-糖链和N-糖肽进行富集,并结合碱性反相色谱进行肽段的预分离和高准确度质谱分析,最终在健康人的血浆中鉴定到了299个糖基化蛋白、637个糖基化位点,并识别出31种不同的糖型。在这些鉴定到的糖基化位点中,新发现有107个N-糖基化位点(占总位点数的16.8%)。本方法操作简单,可以有效富集N-糖肽和N-糖,为在血浆中寻找糖蛋白和糖链生物标志物提供了可靠的手段。

关键词: N-糖, N-糖基化蛋白, 超滤辅助样品制备, 反相色谱, 亲水相互作用色谱

Abstract:

N-glycosylation of proteins is one of the most important post-translational modifications (PTM). Many diagnostic biomarkers and therapeutic targets are glycosylated proteins. However, it is still a big challenge to identify glycoproteins in human plasma, because of the high dynamic range and microheterogeneity of glycosylation. In this work, hydrophilic interaction liquid chromatography (HILIC) enrichment, high-pH reversed-phase prefractionation and high accurate mass spectrometry (MS) analysis were combined to profile the N-glycoproteins in human plasma. In total, 637 N-glycosites from 299 glycoproteins (protein groups) were identified. There were also 31 glycoforms recognized after HILIC enrichment and MS analysis. Among the results, 107 glycosylation sites (16.8%) are newly found. This study provided a simple and reliable strategy for exploring potential N-glycoprotein biomarkers from complicated biological systems.

Key words: N-glycan, N-linked glycoprotein, filter-aided sample preparation (FASP), hydrophilic interaction liquid chromatography (HILIC), reversed-phase liquid chromatography (RPLC)

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