基于Fe_3O_4/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

建立了基于Fe3O4/乙二胺四乙酸（EDTA）磁性粒子的集成化蛋白质组学研究方法。首先用共沉淀法合成EDTA负载的Fe3O4/EDTA磁性粒子。在优化的溶液条件下（95%乙腈-1%三氟乙酸,体积分数）,100μg Fe3O4/EDTA磁性粒子可吸附12.4μg牛血清白蛋白（BSA）,吸附容量是商品化磁珠的10倍左右。以BSA作为标准蛋白质,对所合成的Fe3O4/EDTA磁性粒子作为蛋白质组学反应器的酶解时间进行了优化,发现Fe3O4/EDTA磁性粒子处理BSA酶解1、8和16 h的肽段序列覆盖率和特征肽段结果相当。因此,可以将复杂的蛋白质样品前处理时间缩短至2 h内。最后,将所合成的Fe3O4/EDTA磁性粒子应用于血清的蛋白质组学研究,成功地鉴定出218种蛋白质,其中包含了41种美国食品药品管理局（FDA）认证的生物标志物。... 更多

A Fe3O4/EDTA (ethylenediaminetetraacetic acid) magnetic beads-based integrated method for proteomic analysis was developed. Fe3O4 magnetic beads loaded with EDTA were prepared firstly via co-precipitation method. One hundred tig Fe3O4/EDTA magnetic beads could adsorb 12. 4 mu g BSA (bovine serum albumin) in the optimized solution (95% acetonitrile-1% trifluoracetic acid, v/v). The binding capacity was about 10 times higher than that of the commercialized magnetic beads. The digestion time of Fe3O4/EDTA magnetic beads as the proteomic reactor was also optimized using BSA as the standard protein. The results indicated that the peptide coverage and unique peptides obtained from Fe3O4/EDTA magnetic beads with digestion time of 1, 8 and 16 h were comparable. We then applied the Fe3O4/EDTA magnetic beads for the serum proteome analysis. A total of 218 proteins were identified successfully, including 41 biomarkers approved by Food and Drug Administration (FDA). Protein preconcentration, reduction, alkylation, digestion, peptide desalting, and elution can be achieved in an integrated manner by the Fe3O4/EDTA magnetic beads-based proteomics sample preparation, which reduced the sample loss during sample transfering and processing. The developed method is fast, sensitive and easy to operate, which has prospective application for the clinic proteomics research.