Environmental and Occupational Health Sciences Institute, A Joint Institute of Rutgers University and the University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, NJ 08854, USA.
Anal Biochem. 2011 Feb 1;409(1):138-43. doi: 10.1016/j.ab.2010.10.012. Epub 2010 Oct 13.
Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra-performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2'-deoxyguanosine (2dG) and 5-methyl-2'-deoxycytidine (5mdC) were resolved in less than 1 min with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG, respectively. The accuracy of detection was 95% or higher, and the day-to-day coefficient of variation was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large-scale analysis of epidemiological and clinical DNA samples.
表观遗传修饰，如 DNA 甲基化，在基因表达的转录调控中发挥着关键作用。最近，人们发现几种疾病（包括癌症）以及环境毒物暴露后，全球 DNA 甲基化水平发生了改变。基于全球 DNA 甲基化状态作为疾病状态和环境毒物暴露的生物标志物的潜在用途，我们试图开发一种快速、灵敏和精确的分析方法，用于使用带有离子阱串联质谱检测的超高效液相色谱定量测量全球 DNA 甲基化状态。使用熔合核硅胶柱，在不到 1 分钟的时间内就可以分离 2'-脱氧鸟苷（2dG）和 5-甲基-2'-脱氧胞苷（5mdC），5mdC 和 2dG 的检测限分别为 0.54 和 1.47 fmol。检测的准确性达到 95%或更高，并且发现日间变异系数为 3.8%。该方法通过用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷处理细胞后对全球 DNA 甲基化状态进行定量来验证，该抑制剂将对照细胞中的 DNA 甲基化从 3.1%降低到处理细胞中的 1.1%。该方法的灵敏度和高通量使其适合于对流行病学和临床 DNA 样本进行大规模分析。
