[Simultaneous analysis of global DNA methylation and hydroxymethylation in tissues by liquid chromatography-tandem...

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.