液相色谱-串联质谱法同时测定生物组织全基因组DNA甲基化和羟甲基化水平

Simultaneous analysis of global DNA methylation and hydroxymethylation in tissues byliquid chromatography-tandem mass spectrometry

测定全基因组DNA甲基化和羟甲基化水平对于研究环境污染物暴露的影响及致病机理具有重要的作用。本文建立了液相色谱-串联质谱(LC-MS/MS)同时测定动物组织中全基因组DNA甲基化和羟甲基化水平的方法。从动物组织样品中提取DNA,并将其酶解成单核苷,利用液相色谱-串联质谱法测定5-甲基胞嘧啶核苷、5-羟甲基胞嘧啶核苷和鸟嘌呤核苷的含量,计算全基因组DNA甲基化率和羟甲基化率。利用该方法研究了砷暴露对大鼠肝脏和小脑全基因组DNA甲基化和羟甲基化水平的影响,得到了砷影响DNA甲基化及羟甲基化的初步数据。该方法具有良好的重现性、灵敏度和稳定性,可以同时检测差异较大的DNA甲基化和羟甲基化水平。为同时研究DNA甲基化和羟甲基化水平提供了有力的支持。

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 than0.99.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.