Huang Guang, Sun Zhen, Qin Hongqiang, Zhao Liang, Xiong Zhichao, Peng Xiaojun, Ou Junjie, Zou Hanfa
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
Analyst. 2014 May 7;139(9):2199-206. doi: 10.1039/c4an00076e.
Hydrazide chemistry is a powerful technique in glycopeptides enrichment. However, the low density of the monolayer hydrazine groups on the conventional hydrazine-functionalized magnetic nanoparticles limits the efficiency of glycopeptides enrichment. Herein, a novel magnetic nanoparticle grafted with poly(glycidyl methacrylate) (GMA) brushes was fabricated via reversible addition-fragmentation chain transfer (RAFT) polymerization, and a large amount of hydrazine groups were further introduced to the GMA brushes by ring-opening the epoxy groups with hydrazine hydrate. The resulting magnetic nanoparticles (denoted as Fe3O4@SiO2@GMA-NHNH2) demonstrated the high specificity of capturing glycopeptides from a tryptic digest of the sample comprising a standard non-glycosylated protein bovine serum albumin (BSA) and four standard glycoproteins with a weight ratio of 50 : 1, and the detection limit was as low as 130 fmol. In the analysis of a real complex biological sample, the tryptic digest of hepatocellular carcinoma, 179 glycosites were identified by the Fe3O4@SiO2@GMA-NHNH2 nanoparticles, surpassing that of 68 glycosites by Fe3O4@SiO2-single-NHNH2 (with monolayer hydrazine groups on the surface). It can be expected that the magnetic nanoparticles modified with hydrazine functionalized polymer brushes via RAFT technique will improve the specificity and the binding capacity of glycopeptides from complex samples, and show great potential in the analysis of protein glycosylation in biological samples.
酰肼化学是糖肽富集的一种强大技术。然而，传统酰肼功能化磁性纳米颗粒上单层肼基团的低密度限制了糖肽富集的效率。在此，通过可逆加成-断裂链转移（RAFT）聚合制备了一种接枝有聚甲基丙烯酸缩水甘油酯（GMA）刷的新型磁性纳米颗粒，并用肼水合物使环氧基团开环，从而将大量肼基团进一步引入到GMA刷中。所得磁性纳米颗粒（表示为Fe3O4@SiO2@GMA-NHNH2）从包含标准非糖基化蛋白质牛血清白蛋白（BSA）和四种标准糖蛋白且重量比为50∶1的样品的胰蛋白酶消化物中捕获糖肽时表现出高特异性，检测限低至130 fmol。在实际复杂生物样品即肝癌细胞胰蛋白酶消化物的分析中，Fe3O4@SiO2@GMA-NHNH2纳米颗粒鉴定出179个糖基化位点，超过了Fe3O4@SiO2-单-NHNH2（表面具有单层肼基团）的68个糖基化位点。可以预期，通过RAFT技术用酰肼功能化聚合物刷修饰的磁性纳米颗粒将提高从复杂样品中捕获糖肽的特异性和结合能力，并在生物样品中蛋白质糖基化分析中显示出巨大潜力。
