色谱

色谱 ›› 2015, Vol. 33 ›› Issue (3): 221-227.DOI: 10.3724/SP.J.1123.2014.11022

• 研究论文 • 上一篇    下一篇

新型核壳型亲水聚合物-硅胶杂化填料的合成及其在蛋白质N-糖链富集中的应用

白海红1,2, 范超2, 沈丙权1,2, 田芳2, 邓玉林1, 潘一廷3, 秦伟捷2, 钱小红2   

  1. 1. 北京理工大学生命学院, 北京 100081;
    2. 军事医学科学院放射与辐射医学研究所, 北京蛋白质组研究 中心, 蛋白质组学国家重点实验室, 北京 102206;
    3. 北京石油化工学院化学工程学院, 北京 102617
  • 收稿日期:2014-11-16 修回日期:2014-12-11 出版日期:2015-03-08 发布日期:2015-02-12
  • 通讯作者: 潘一廷, 秦伟捷
  • 基金资助:

    国家重大科学计划项目(2013CB911204);国家重大科学仪器设备开发专项项目(2011YQ09000504);国家自然科学基金项目(21275005,21235001).

Synthesis of core-shell hydrophilic polymer-silica hybrid material and its application in N-glycan enrichment

BAI Haihong1,2, FAN Chao2, SHEN Bingquan1,2, TIAN Fang2, DENG Yulin1, PAN Yiting3, QIN Weijie2, QIAN Xiaohong2   

  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 Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, China
  • Received:2014-11-16 Revised:2014-12-11 Online:2015-03-08 Published:2015-02-12

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摘要：

蛋白质的N-糖基化修饰与多种重要的生理、病理进程密切相关,是多种重大疾病诊断标志物研究的热点。由于糖蛋白本身多是低丰度表达的蛋白质,且糖链结构具有高度微不均一性,这使得蛋白质糖基化修饰的分析具有一定的挑战。本研究利用表面引发-原子转移自由基聚合(SI-ATRP)法,以带有双键的氨基葡萄糖为单体(GMAG),成功制备了新型核壳型亲水聚合物-硅胶杂化填料(pGMAG-SiO2)。由于在硅胶表面引入致密的亲水聚合物层,该填料不仅保持了硅胶良好的机械强度,而且显著提高了其亲水性,因此非常适合作为亲水填料用于蛋白质的N-糖链富集。以麦芽七糖和鸡卵清蛋白的N-糖链为研究对象,考察了该填料对N-糖链的富集效果,并将该杂化填料成功用于人血浆中糖蛋白N-糖链的富集检测,共鉴定了47种糖型。以上结果表明,pGMAG-SiO2填料对N-糖链具有较高的亲和性,可以用于N-糖链的高覆盖率鉴定。

关键词: N-糖基化蛋白质, N-糖链, 表面引发-原子转移自由基聚合反应, 核壳型硅胶填料

Abstract:

Protein N-glycosylation is one of the most important post-translational modifications closely correlated with many important biological and pathological processes. The structural alterations of N-linked glycans in glycoproteins are always associated with many diseases, such as diabetes, heart failure and malignant tumors. Therefore, it is very important to establish sensitive methods for high-throughput N-glycan profiling. However, the low abundance of the N-glycoproteins and the heterogeneity of the N-glycans make it a challenge to analyse the protein glycosylation sensitively. In this work, we had synthesized core-shell hydrophilic polymer-silica hybrid materials (pGMAG-SiO2) for the efficient enrichment of protein N-glycans. Firstly, pGMAG-SiO2 was prepared by in situ growth of glucose polymer on the surface of silica microparticles using surface-initiated atom transfer radical polymerization (SI-ATRP) technique. The strong hydrophilicity of the material makes it suitable for the enrichment of N-glycans released from complex samples. Secondly, maltoheptaose and the N-glycans from chicken egg albumin were used as standard samples to optimize the enrichment conditions and evaluate the enrichment efficiency of pGMAG-SiO2. Finally, pGMAG-SiO2 was applied to the enrichment of N-linked glycans from human plasma proteins and 47 glycoforms were successfully identified after enrichment. These results demonstrated the high enrichment efficiency and significant application value of pGMAG-SiO2 in the analysis of N-glycans.

Key words: N-glycan, N-glycoprotein, core-shell silica packing, surface-initiated atom transfer radical polymerization (SI-ATRP)

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