高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立

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。

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

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.