组织中全基因组DNA甲基化的液相色谱-串联质谱分析  

Analysis of Global DNA Methylation in Tissue by Liquid Chromatography-Tandem Mass Spectrometry

作　　者：张俊杰[1] 张立坚[1] 刘春安[1] 张良滔[1] 蔡春[1] 

机构地区：[1]广东医学院,广东湛江524023

出　　处：《质谱学报》2010年第6期326-330,共5页Journal of Chinese Mass Spectrometry Society

摘　　要：建立液相色谱-串联质谱测定组织中全基因组DNA甲基化水平的方法。采用苯酚氯仿提取组织DNA,提取的DNA用88%甲酸在140℃下裂解,DNA裂解液加入同位素胞嘧啶作内标,经N2吹干后,用甲醇溶解,以液相色谱-串联质谱检测胞嘧啶和5-甲基胞嘧啶的含量,并计算全基因组中DNA甲基化的水平。结果表明,胞嘧啶的线性范围为1～100μg·L^-1相关系数为0.997 4,相对标准偏差为0.70%～4.09%;5-甲基胞嘧啶的线性范围为1～50μg·L^-1相关系数为0.994 8,相对标准偏差为0.60%～4.81%。胞嘧啶和5-甲基胞嘧啶的检出限为1 pg,日内相对标准偏差为1.86%～4.67%,日间相对标准偏差为3.72%～4.68%,胞嘧啶和5-甲基胞嘧啶的加样回收率为86.52%～105.14%。本研究所建立的方法检测组织中DNA甲基化程度,具有专一性强、操作简便的优点,能较好的满足全基因组DNA甲基化检测的要求。Global DNA methylation in tissue was determined by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.DNA was extracted by phenol-chloroform,hydrolyzed using 88% formic acid at 140 ℃,spiked with cytosine-2,4-13C2,15N2 as internal standard,reconstituted in methanol and analyzed by LC-MS/MS with multiple reaction monitoring mode,to reflect the global DNA methylation level of the tissue.Results show that the limit of quantification is 1 μg·L^-1or both Cytosine（Cyt） and 5-methylcytosine（5mCyt）,and the linear ranges of calibration curve are 1—50 μg·L^-1nd 1—100 μg·L^-1or 5mCyt and Cyt,respectively,with correlation coefficient higher than 0.99.The relative standard deviations（RSDs） are 0.70%—4.09% and 0.60%—4.81% for Cyt and 5mCyt,respectively.The intra-day precision expressed as RSD ranges from 1.86% to 4.67%,while the inter-day values from 3.72% to 4.68%.The recovery ratio of method varies from 86.52% to 105.14%.The method is simple,and can be used for detection of Cyt and 5mCyt,thus enabling the evaluation of global DNA methylation.

关 键 词：DNA甲基化 液相色谱-串联质谱(LC-MS/MS) 胞嘧啶 5-甲基胞嘧啶 

分 类 号：O657.63[理学—分析化学]