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

Abstract

Measuring global DNA methylation and hydroxymethylation is important in resear-ches of effect and mechanism of environmental pollutants exposure. A method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was devel-oped to simultaneously determine global DNA methylation and hydroxymethylation level in bio-logical 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-hydroxymethylcy-tosine , and were 0. 2 and 0. 6 ng/mL for deoxyguanosine. The developed method was further successfully appl ed to nvest gate global DNA methylat on and hydroxymethylat on alterat on n liver and cerebellum of rats exposed to arsenic via drinking water. This approach could quanti-tat vely detect 5-methylcyt d ne, 5-hydroxymethylcytos ne and deoxyguanos ne w th h gh sens-t v ty, repeatab l ty and stab l ty. Our study prov ded a means to s multaneously analyze global DNA methylat on and hydroxymethylation.