Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry

Promoter hypermethylation associated tumor suppressor genes (TSGs) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient’s clinical response. Herein, a LC-MS/MS assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2′-deoxycytidine (5mdC) to 2′-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted PCR amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intra-day precision ranging from 3.00 to 16.0% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis, which could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.