高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立 |
| |
引用本文: | 张航,;胡俊杰,;汤瑞华,;叶金波,;任晓虎,;胡韬,;黄培武,;杨细飞,;黄海燕,;刘建军. 高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立[J]. 生物技术通讯, 2014, 0(4): 537-541 |
| |
作者姓名: | 张航, 胡俊杰, 汤瑞华, 叶金波, 任晓虎, 胡韬, 黄培武, 杨细飞, 黄海燕, 刘建军 |
| |
作者单位: | [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 |
| |
基金项目: | 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。
|
关 键 词: | 高效液相色谱 串联质谱 DNA甲基化 |
Establishment of HPLC Coupled with Tandem Mass Spectrometry for Genome-Wide DNA Methylation Detection |
| |
Affiliation: | ZHANG Hang,HU Jun-Jie,TANG Rui-Hua,YE Jin-Bo,REN Xiao-Hu,HU Tao,HUANG Pei-Wu,YANG Xi-Fei,HUANG Hai-Yan,LIU Jian-Jun(1. College of Life Science, Shenzhen University, Shenzhen 518060; 2. Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055; China) |
| |
Abstract: | 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. |
| |
Keywords: | high performance liquid chromatography tandem mass spectrometry DNA methylation |
本文献已被 CNKI 维普 等数据库收录! |
|