液相色谱-串联质谱法检测甲基化和乙酰化修饰的组蛋白H3及其在表观遗传药物评估中的应用  

Detection of Histone H3 Methylation and Acetylation Modifications by Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Epigenetic Drug Evaluation

作　　者：史钦鋆 瞿敏敏 李治 马波 陈佳 徐斌 徐华 谢剑炜 SHI Qin-Yun;QU Min-Min;LI Zhi;MA Bo;CHEN Jia;XU Bin;XU Hua;XIE Jian-Wei(School of Pharmacy,Qingdao University,Qingdao 266000,China;National Key Laboratory of National Security Special Drugs,Beijing 100850,China)

机构地区：[1]青岛大学药学院,青岛266000 [2]军事医学研究院国家安全特需药品全国重点实验室,北京100850

出　　处：《分析化学》2024年第6期818-827,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21974151)资助。

摘　　要：基于液相色谱-串联质谱技术(Liquid chromatography tandem mass spectrometry,LC-MS/MS),采用三重四极杆质谱的多反应选择离子监测(Multiple reaction monitoring,MRM)模式,应用自下而上的蛋白分析策略,对细胞组蛋白H3 N端的常见甲基化和乙酰化修饰位点及表达水平进行定量分析。将本方法应用于28种表观遗传药物引起的细胞组蛋白H3修饰变化的检测,结果表明,其中25种药物(包括去乙酰化酶抑制剂Abexinostat、Valproic acid和AGK7等)暴露HepG2细胞24 h后诱导的组蛋白H3修饰变化与其已报道的生物活性一致,同时也可检测到其它修饰变化;其余3种药物,包括去甲基化酶抑制剂IOX1、GSK-j1和乙酰化转移酶抑制剂L002,在暴露细胞后仅检测到与已报道活性不同的修饰变化;总体检测吻合率达到89.3%。本方法能通量化定量分析组蛋白H3修饰位点及其变化,具有特异性好和修饰信息丰富等优势,有望为表观遗传活性化合物的筛选评估及作用机制探讨提供新的技术工具。The changes in epigenetic modifications of histones are one of the important factors in cancer development and metastasis,and the development of epigenetic therapies for cancer treatment has led to epigenetic drug screening as a research focus.In this work,the common methylation and acetylation modifications at the N-terminal of cellular histones H3 were quantified by a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method,and a throughput assay for screening and assessment of epigenetic drug was established.A total of 39 kinds of modification combinations containing common methylation and acetylation sites of H3 peptides were simultaneously monitored by triple quadrupole mass spectrometry in multiple reaction monitoring(MRM)mode.The developed method was applied to analyze HepG2 cells exposed for 24 h to 28 kinds of epigenetic drugs that could modulate the level of methylation or acetylation modifications.Results showed that 25 of these drugs,such as deacetylase inhibitors Abexinostat,Valproic acid and AGK7,induced histone H3 modification changes in the exposed cells that were consistent with those reported in the literature,while other modification changes were also detectable.Three of these drugs,including demethylase inhibitors IOX1,GSK-j1 and acetyltransferase inhibitor L002,however,induced modification changes different from those reported in the literature.An overall test match rate of 89.3%was achieved.The established LC-MS/MS method could quantitatively analyze histone H3 modification sites and their changes in cells in a high-throughput and highly sensitive manner,and could be applied to the evaluation of epigenetic drugs with known activities,with good specificity and rich modification information,which was expected to provide a new technological tool for screening and evaluation of epigenetically active compounds and exploration of their mechanism of action.

关 键 词：表观遗传药物 液相色谱-串联质谱 组蛋白 甲基化 乙酰化 

分 类 号：R96[医药卫生—药理学] O657.63[医药卫生—药学]