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

Zhongfa Liu, Jiejun Wu, Zhiliang Xie, Shujun Liu, Patty Fan-Havard, Tim H.M. Huang, Christoph Plass, Guido Marcucci, Kenneth K. Chan

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


Promoter hypermethylation-associated tumor suppressor gene (TSG) 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. Here a liquid chromatography-tandem mass spectrometry (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 polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% 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 that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.

Original languageEnglish (US)
Pages (from-to)106-113
Number of pages8
JournalAnalytical Biochemistry
Issue number2
StatePublished - Aug 15 2009
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported in part by the National Cancer Institute (Bethesda, MD, Grant CA102031) and the Biomedical Mass Spectrometry Facility at The Ohio State University.

Copyright 2020 Elsevier B.V., All rights reserved.


  • LC-MS/MS
  • Quantification
  • Regional DNA methylation

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