色谱 ›› 2014, Vol. 32 ›› Issue (4): 369-375.DOI: 10.3724/SP.J.1123.2013.12018

• 庆祝《色谱》创刊三十周年暨卢佩章院士九十华诞专刊-研究论文 • 上一篇    下一篇

伴刀豆凝集素修饰磁性纳米粒子富集人血清中糖蛋白及质谱鉴定

李凤, 康经武   

  1. 中国科学院上海有机化学研究所, 上海 200032
  • 收稿日期:2013-12-18 修回日期:2014-01-08 出版日期:2014-04-08 发布日期:2014-03-28
  • 通讯作者: 康经武
  • 基金资助:

    国家自然科学基金项目(21175146,20975109).

Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry

LI Feng, KANG Jingwu   

  1. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
  • Received:2013-12-18 Revised:2014-01-08 Online:2014-04-08 Published:2014-03-28

摘要:

发展了一种新型的磁性纳米粒子应用于人血清中特异性糖蛋白的亲和富集。制备的磁性纳米粒子具有核/壳/壳结构,即由Fe3O4磁性粒子/硅胶层/有机聚合物外层构成。伴刀豆凝集素A(Con A)以共价键合的形式通过短链聚乙二醇固定在粒子表面,实现了人血清中特异性糖蛋白的高效富集。富集的蛋白经过胰蛋白酶酶解后,所得的肽段经离线的二维色谱分离,用高分辨质谱共鉴定出80种蛋白。通过NetNGlyc等搜索软件分析确定其中76种为糖蛋白,分析发现在血清中质量浓度仅为0.00001 g/L的 β -2-glycoprotein 1也得到了鉴定,表明我们发展的磁性纳米粒子与凝集素相结合的方式,可以高效地富集复杂体系中与主要蛋白成分含量相差12个数量级的低丰度糖蛋白。

关键词: 磁性纳米粒子, 富集, 凝集素, 糖蛋白, 液相色谱, 质谱

Abstract:

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP@ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. β-2-Glycoprotein 1 present in serum at low mass concentration around 0.00001 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP@ConA has been proven to be an efficient alternative to currently available immobilization supports.

Key words: enrichment, glycoprotein, lectin, liquid chromatography (LC), magnetic nanoparticle, mass spectrometry (MS)

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