用于质谱分析的细胞总蛋白三种酶切方法比较  

Comparison of preparation of total cell protein sample for MS analysis by three digestion methods

作　　者：胡家 杨颖 李蕾 殷爱红 李慎涛 HU Jia;YANG Ying;LI Lei;YIN Aihong;LI Shentao(Central Laboratory,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学中心实验室

出　　处：《山西医科大学学报》2019年第6期848-853,共6页Journal of Shanxi Medical University

基　　金：北京市教育委员会科技计划面上项目(M201510025004)

摘　　要：目的比较无去污剂的细胞总蛋白三种酶切样品制备方法。方法采用细胞吸入胶内酶切法、蛋白吸入胶内酶切法及超滤管辅助溶液酶切法对细胞总蛋白进行酶切,再用液质联用技术对三种酶切方法制备的多肽样品进行蛋白鉴定。结果通过液质联用技术鉴定,细胞吸入胶内酶切法制备的样品获得3835个蛋白,蛋白吸入胶内酶切法制备的样品获得2340个蛋白,超滤管辅助溶液酶切法制备的样品获得4248个蛋白。韦恩图分析显示,不同酶切方法得到的蛋白种类存在差异。对细胞吸入胶内酶切法和超滤管辅助溶液酶切法所获得的蛋白进行基因本体(geneontology,GO)分析发现,细胞吸入胶内酶切法能获得更多膜结合蛋白。结论三种无去污剂酶切方法,避免了去污剂的种种弊端,其中超滤管辅助溶液酶切法和细胞吸入胶内酶切法更优。Objective To compare the total cell protein samples without detergent used in MS analysis prepared by the three digestion methods.Methods The total cell protein samples were digested by the cell absorption-based in-gel digestion method,the protein absorption-based in-gel digestion method or filter-aided sample preparation(FASP)method,respectively.The total cell proteins of the samples prepared by the three digestion methods were analyzed by liquid chromatography with mass spectrometry(LC-MS).Results A total of 3 835 proteins were identified from the sample prepared by the cell absorption-based in-gel digestion method,2 340 proteins from the sample prepared by the protein absorption-based in-gel digestion method,and 4 248 proteins from the sample prepared by FASP.The Venn diagram analysis showed that there was different types of proteins identified by different methods.GO analysis of proteins from the cell absorption-based in-gel digestion method and FASP method showed that more membrane binding proteins were identified by the cell absorption-based in-gel digestion method.Conclusion The three digestion methods without detergent could avoid the negative influence of the detergent on MS analysis.Of the three methods used,FASP and the cell absorption-based in-gel digestion method are better.

关 键 词：细胞吸入胶内酶切法 蛋白吸入胶内酶切法 超滤管辅助溶液酶切法 质谱分析 

分 类 号：Q503[生物学—生物化学]