Quantification of DNA Methylation States

Genomic DNA was isolated by Phenol:chloroform method. 5 μg of genomic DNA was digested to nucleoside level by using Nucleoside digestion mix (M0649S; NEB). Mass spectrometry-based quantitation of cytosine, 5mC and 5hmC was performed as described (59 (link), 60 (link)). Briefly, Cytosine, 5mC, 5hmC were quantitated using a Thermo Vanquish UHPLC coupled to a Thermo TSQ-Altis tandem mass spectrometer through an electrospray ion source operating in positive ion mode at 3.5kV. Stock standard solutions were prepared in deionized water at a concentration of 1 mmol/L each. Calibration standard mixtures were prepared at concentrations between 1.0–250umol/L for dC, 0.04–10 umol/L for mdC, and 0.002–0.5 umol/L for hmdC. Linear calibration plots were prepared using concentration versus peak area integration (zero intercept) with a R² greater than 0.999. By comparing the internal standard normalized peak areas in the digest sample to the corresponding retention times from the calibration standards, the micromolar concentrations of the nucleosides were determined against the standard curve. The molar ratio as a percent was then calculated as follows:
$$\underset{\_}{\text{Mol}\%\text{hmdC}}=(\left[\text{hmdC}\right]/\left(\left[\text{dC}\right]+\left[\text{mdC}\right]+\left[\text{hmdC}\right]\right)\times 100$$