高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立  

Establishment of HPLC Coupled with Tandem Mass Spectrometry for Genome-Wide DNA Methylation Detection

作　　者：张航[1,2] 胡俊杰[2] 汤瑞华[2] 叶金波[2] 任晓虎[2] 胡韬[2] 黄培武[1,2] 杨细飞[2] 黄海燕[2] 刘建军[2] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060 [2]深圳市疾病预防控制中心现代毒理学重点实验室,广东深圳518055

出　　处：《生物技术通讯》2014年第4期537-541,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81273126);广东省自然科学基金重点项目(S2012020010903);深圳市科技计划重点项目(201201027);深圳市基础研究计划(JCYJ20120616154222545)

摘　　要：目的：建立高效液相色谱-串联质谱（HPLC-ESI-MS/MS）检测基因组DNA甲基化水平的方法。方法：以5-mdC和dG为标准品，采用全自动高效液相色谱系统进行分离，串联电喷雾质谱检测，选择多反应监测模式（MRM）测定标准品，绘制标准工作曲线。结果：在MRM模式下选取5-mdC（m/z 241.9→126.3）和dG（m/z 268.1→152.3）分别作为定量检测的母子离子对，各化合物能实现良好的基线分离；5-mdC和dG碰撞能均为15 eV，去簇电压分别为40和45 V，最低定量限分别为1.65和2.47 fmol；标准品的响应值比为90%~110%；5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论：HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。Objective： To estabilish high performance liquid chromatography（HPLC） coupled with tandem mass spectrometry（HPLC-ESI-MS/MS） for genome-wide DNA methylation detection. Methods： 5-mdC and dG were used as the standard. HPLC system and electronic spray were used to separate and detect the standard at multi-ple reaction monitoring（MRM） mode; then the standard working curve was generated by the data obtained. Re-sults： The 5-mdC（m/z 241.9→126.3） and dG（m/z 268.1→152.3） were chosen as parent and child ion pairs for quantitative detection at MRM mode. A good baseline separation can be achieved for the compounds. The collision energy of both 5-mdC and dG was 15 eV, and the cluster voltage was 40 and 45 V, respectively. The DNA meth-ylation rate of 5-mdC and dG was 1% under the minimum quantitative limit. The limit of quantitation of 5-mdC and dG was 1.65 and 2.47 fmol, respectively. The response values of 5-mdC and dG standards range between 90% and 110%. The intra- and inter-day precisions（RSD） of 5-mdC were less than 8%. Conclusion： HPLC-ESI-MS/MS with high-throughput, high sensitivity, high accuracy and good repeatability can be used to measure DNA methylation levels.

关 键 词：高效液相色谱 串联质谱 DNA甲基化 

分 类 号：Q503[生物学—生物化学]