利用亲和层析法检测乙酰化蛋白的研究  

Detecting acetylated proteins by affinity chromatography column

作　　者：郑力[1] 钟艳平[1] 萧浩[1] 周怡[1] 罗蓉[1] 李洪涛[1] 李刚[1] 廖明[1] 何敏[1] 

机构地区：[1]广西医科大学医学科学实验中心,南宁530021

出　　处：《中华血液学杂志》2012年第3期211-214,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金（30960332）;广西科学研究与技术开发计划项目（桂科能0630006-5E、桂科能0842009）;广西医科大学医学科学实验中心开放基金（FJJ201045）

摘　　要：目的建立一种快速、相对定量检测乙酰化蛋白方法，并初步应用其检测药物作用后Jurkat细胞乙酰化总蛋白水平。方法用不同浓度的曲古菌素A（TSA）诱导Jurkat细胞蛋白高乙酰化后，利用抗乙酰化赖氨酸抗体亲和层析柱富集纯化乙酰化总蛋白，经酸洗脱后固定于酶标板，ELISA法检测乙酰化蛋白相对总量，并用基质辅助激光解吸电离串联飞行时间质谱仪（MALDI—TOF／TOF）分析验证其成分；同法检测不同浓度的没食子酸、大黄素、单乙酰化大黄素A三种药物作用Jurkat细胞后乙酰化总蛋白水平的变化，筛查诱导Jurkat细胞蛋白乙酰化药物。结果1μmol／LTSA作用于4×10^5Jurkat细胞24h，乙酰化总蛋白相对水平最高。MALDI—TOF／TOF分析显示，TSA诱导Jurkat细胞产生的乙酰化蛋白共有22种，其中15种为乙酰化组蛋白。没食子酸、大黄素、单乙酰化大黄素A作用Jut-kat细胞后所导致的乙酰化程度不同，以1μmol／L浓度TSA为阳性对照组，无药物为空白对照组，35．09μmol／L和17．54μmol／L大黄素处理的Jurkat细胞乙酰化蛋白相对水平分别为4．3％和14．2％；1．47μmol／L和2．94μmol／L没食子酸处理组乙酰化蛋白相对水平分别为28．7％和11．5％；152．91μmol／L和30．58μmol／L单乙酰化大黄素组分别为22．O％和3．6％；其中1．47μmol／L没食子酸所诱导的乙酰化蛋白水平最高。结论初步建立了活细胞基础上纯化富集并检测乙酰化总蛋白水平的方法，该法可快速、简便的筛选以组蛋白去乙酰化酶为靶点的抗癌药物。Objective To establish a rapid, relatively quantitative method of detecting acetylated proteins. Methods The proteins of Jurkat cells were acetylated by Trichostatin A （TSA） at different concentrations, then enriched and purified by anti-acetylated lysine antibodies affinity chromatography colum. The components eluted by acid were fixed on the mieroplate, the levels of aeetylated proteins were tested by ELISA, and their components were identified by MALDI-TOF-TOF mass spectrometry. Also the above-mentioned methods were applied to the other three agents （gallic acid, emodin and monoacetylated emodin A）. Results That 4 ×10^5 Jurkat ceils treated with 1 μmol/L TSA produced the optimal acetylated effect, up to 22 acetylated proteins were identified by MALDI-TOF-TOF, of them 15 were acetylated histones. The other three agents also induced acetylation, the relative values of acetylated proteins of Jurkat cells treated with 35.09 μmol/L and 17.54 μmol/L gallic acid were 4.3% and 14.2% respectively; those as of 28.7% and 11.5% treated with 1.47 μmol/L and 2.94 izmol/L emodin; those as of 22.0% and 3.6% treated with 152.91 μmol/L and 30.58 μmol/L monoacetylated emodin A. Conclusion The method based on affinity chromatography colum may be useful for the detection of acytylated proteins, and could be used to screen agents which target to histone deacetylase.

关 键 词：组蛋白类 乙酰化作用 曲古菌素A 色谱法 亲和 细胞系 JURKAT 

分 类 号：R446.1[医药卫生—诊断学]