Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan.
Anal Bioanal Chem. 2013 Nov;405(27):8859-69. doi: 10.1007/s00216-013-7305-3. Epub 2013 Aug 25.
Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.
全球 DNA 甲基化分析为生物学研究提供了重要的见解，并揭示了 DNA 甲基化的广泛作用。我们描述了在线固相萃取和同位素稀释液相色谱/串联质谱（LC-MS/MS）联用技术，用于同时测量 DNA 中的 5-甲基-2'-脱氧胞苷（5-medC）和 2'-脱氧胞苷（dC）。通过引入同位素内标和在线富集技术，该方法的检测限估计低至 0.065pg，仅需皮克级的 DNA 即可检测到人类全基因组 DNA 甲基化。该方法用于评估在准确测定全基因组 DNA 甲基化水平和保证 DNA 完全消化所需的最佳酶用量，并用于检测 79 例肺癌患者肿瘤旁肺组织的全基因组甲基化水平。我们进一步通过 LC-MS/MS 检测了肺癌患者的甲基化（N7-甲基鸟嘌呤（N7-meG）、O（6）-甲基鸟嘌呤（O（6）-meG）和 N3-甲基腺嘌呤（N3-meA））和氧化 DNA 损伤（8-氧代-7,8-二氢-2'-脱氧鸟苷（8-oxodG））。优化实验表明，在 DNA 中，核酸酶 P1 和碱性磷酸酶（AP）从 DNA 中释放 dC 的速度比 5-medC 快得多，如果酶消化不充分，可能会导致全基因组 DNA 甲基化测量出现误差。核酸酶 P1 对 5-medC 和 dC 的活性差异比 AP 更大。腺癌和鳞癌患者的全基因组 DNA 甲基化水平相似，范围在 3.16-4.01%。吸烟和性别对全基因组 DNA 甲基化水平没有影响，也与肺癌患者的 N7-meG 或 8-oxodG 无相关性。然而，在所有肺组织样本中均未检测到 O（6）-meG 和 N3-meA 的水平。
