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

doi:10.1016/j.ab.2009.05.012

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 journal › Article › peer-review

24 Scopus citations

Abstract

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 language	English (US)
Pages (from-to)	106-113
Number of pages	8
Journal	Analytical Biochemistry
Volume	391
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2009.05.012
State	Published - Aug 15 2009
Externally published	Yes

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.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

LC-MS/MS
Quantification
Regional DNA methylation

Publisher link

10.1016/j.ab.2009.05.012

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title = "Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry",

abstract = "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.",

keywords = "LC-MS/MS, Quantification, Regional DNA methylation",

author = "Zhongfa Liu and Jiejun Wu and Zhiliang Xie and Shujun Liu and Patty Fan-Havard and Huang, \{Tim H.M.\} and Christoph Plass and Guido Marcucci and Chan, \{Kenneth K.\}",

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. ",

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doi = "10.1016/j.ab.2009.05.012",

language = "English (US)",

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AU - Liu, Zhongfa

AU - Wu, Jiejun

AU - Xie, Zhiliang

AU - Liu, Shujun

AU - Fan-Havard, Patty

AU - Huang, Tim H.M.

AU - Plass, Christoph

AU - Marcucci, Guido

AU - Chan, Kenneth K.

N1 - 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.

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N2 - 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.

AB - 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.

KW - LC-MS/MS

KW - Quantification

KW - Regional DNA methylation

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ER -

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

Abstract

Bibliographical note

UN SDGs

Keywords

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