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

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

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0.000 01 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。

Biomedical sciences,and in particular biomarker research,demand efficient glyco-protein 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 com-posed 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 poly-ethylene hydrophilic linker,it made the surfaces of the magnetic nanoparticles highly hydrophil-ic 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 immobi-lization of concanavalin A（ MNP@ ConA）. The enriched proteins were reduced,alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chroma-tography. Protein identification was realized with nano-high performance liquid chromatogra-phy-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 ser-um at low mass concentration around 0. 000 01 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.