肝癌细胞分泌蛋白质的多种提取方法比较  

Comparison of Alternative Extraction Methods for Secretome Profiling in Human Hepatocellular Carcinoma Cells

作　　者：曹晶[1] 沈诚频[1] 张军[1] 姚鋆[1] 申华莉[1] 刘银坤[1,2] 陆豪杰[1] 杨芃原[1] 

机构地区：[1]复旦大学化学系/生物医学研究院,上海200032 [2]复旦大学附属中山医院肝癌研究所,上海200032

出　　处：《中国科学：生命科学》2010年第9期853-858,共6页Scientia Sinica(Vitae)

基　　金：国家高技术研究发展计划(批准号:2006AA02A308)资助项目

摘　　要：由于分泌蛋白在重大生理和病理过程中的重要角色和功能,使得分泌蛋白质组的研究备受人们关注.高效提取分泌蛋白质是分泌蛋白质组学研究的第一步,也是关键的一步.本研究首次比较了3种常用的提取方法:超滤法、沉淀法和透析法.以具有高自发性转移潜能的人肝癌细胞系LM3为研究对象,采用3种方法平行提取分泌蛋白,发现超滤法的提取效率最高;蛋白质经过溶液酶解,在线的一维反相高效液相色谱分离和ESI-MS/MS质谱鉴定,共鉴定了360个非冗余蛋白,其中有34,110和29个蛋白分别只在超滤法、沉淀法和透析法提取的样品中被鉴定到,沉淀法占优势,3种方法互相补充.360个蛋白中,有42个分泌蛋白为首次被鉴定到,扩大了已有的人肝癌转移细胞分泌蛋白数据库.Given the importance of secreted proteins as a source for early detection and diagnosis of disease, secreted proteins have been arousing considerable attention. One of the difficulties in secretome study is to extract proteins at low concentrations from a large volume of culture media, which always are rich in salts and other compounds interfering with most proteomics techniques. Ultrafiltration, precipitation and dialysis are three major extraction methods. In this study, it is the first time to compare the three mainly used methods without bias, the merits and shortcomings of each method were discussed based on the obtained results. As a result, centrifugal ultrafiltration method represented the best in extracting efficiency, precipitation method obtained the highest number of confidential identification, and these three methods gave us closely related but different information of secretome, and so they are considered complementary or at least supplementary methods at present. Totally, 360 unique proteins were identified, including 211 secreted proteins potentially. Compared to previous studies, there were also 42 secreted proteins newly identified in this study, thus, this study not only offered a reference for the selection of secretome extraction methods but also expanded the secretome database for the investigation of HCC.

关 键 词：肝癌 分泌蛋白 蛋白提取 超滤 沉淀 透析LC—MS/MS 

分 类 号：R735.7[医药卫生—肿瘤]