Intensive optimization and evaluation of global DNA methylation quantification using LC-MS/MS

Abstract

DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.