Mu Xiaoli, Zhang Jie, Peng Siyuan, Wang Xiaoxue, Shen Heqing
Se Pu. 2014 Jul;32(7):682-6. doi: 10.3724/sp.j.1123.2014.03025.
Measuring global DNA methylation and hydroxymethylation is important in researches of effect and mechanism of environmental pollutants exposure. A method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed to simultaneously determine global DNA methylation and hydroxymethylation level in biological tissues. DNA was extracted from tissues and converted into single nucleotide via enzyme digestion. Liquid chromatography coupled to tandem mass spectrometry was used to measure the concentrations of 5-methylcytidine, 5-hydroxymethylcytosine and deoxyguanosine, which were used to calculate global DNA methylation ratios and hydroxymethylation ratios. The results showed that the correlation coefficients were higher than 0.99. The limit of detection ( LOD, S/N = 3) and the limit of quantification (LOQ, S/N = 10) of 5-methylcytidine were 0.015 and 0.045 ng/mL, respectively. They reached 0.001 and 0.003 ng/mL for 5-hydroxymethylcytosine, and were 0.2 and 0.6 ng/mL for deoxyguanosine. The developed method was further successfully applied to investigate global DNA methylation and hydroxymethylation alteration in liver and cerebellum of rats exposed to arsenic via drinking water. This approach could quantitatively detect 5-methylcytidine, 5-hydroxymethylcytosine and deoxyguanosine with high sensitivity, repeatability and stability. Our study provided a means to simultaneously analyze global DNA methylation and hydroxymethylation.
测量全球DNA甲基化和羟甲基化在环境污染物暴露的影响及机制研究中具有重要意义。本文建立了一种基于液相色谱-电喷雾电离串联质谱（LC-MS/MS）的方法，用于同时测定生物组织中的全球DNA甲基化和羟甲基化水平。从组织中提取DNA，并通过酶消化将其转化为单核苷酸。采用液相色谱-串联质谱法测定5-甲基胞嘧啶、5-羟甲基胞嘧啶和脱氧鸟苷的浓度，用于计算全球DNA甲基化率和羟甲基化率。结果表明，相关系数高于0.99。5-甲基胞嘧啶的检测限（LOD，S/N = 3）和定量限（LOQ，S/N = 10）分别为0.015和0.045 ng/mL。5-羟甲基胞嘧啶的检测限和定量限分别为0.001和0.003 ng/mL，脱氧鸟苷的检测限和定量限分别为0.2和0.6 ng/mL。所建立的方法进一步成功应用于研究经饮水暴露于砷的大鼠肝脏和小脑中全球DNA甲基化和羟甲基化的变化。该方法能够高灵敏度、高重复性和高稳定性地定量检测5-甲基胞嘧啶、5-羟甲基胞嘧啶和脱氧鸟苷。我们的研究提供了一种同时分析全球DNA甲基化和羟甲基化的方法。
