Simultaneous quantification of methylated purines in DNAby isotope dilution LC-MS/MS coupled with automatedsolid-phase extraction

中山醫學大學機構典藏 CSMUIR > 健康管理學院 > 公共衛生學系暨碩士班 > 期刊論文 > Item 310902500/11922

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题名:	Simultaneous quantification of methylated purines in DNAby isotope dilution LC-MS/MS coupled with automatedsolid-phase extraction
作者:	Hu CW;Chen CM;Ho HH;Chao MR
贡献者:	中山醫學大學
关键词:	Online solid-phase extraction;LC-MS/MS;Methylated DNA;Methyl methanesulfonate
日期:	2012-01
上传时间:	2015-08-04T10:23:51Z (UTC)
摘要:	Since methylation at the N-7 and O(6) positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N(7)-methylguanine (N(7)-MeG), O(6)-methylguanine (O(6)-MeG), and N(3)-methyladenine (N(3)-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure N(7)-MeG, O(6)-MeG, and N(3)-MeA in DNA hydrolysates. With the use of isotope internal standards ((15)N(5)-N(7)-MeG, d(3)-O(6)-MeG, and d(3)-N(3)-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N(7)-MeG, O(6)-MeG, and N(3)-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6-9.6% and 2.7-13.6%, respectively. Mean recoveries were 96-109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N(7)-MeG, O(6)-MeG, and N(3)-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N(7)-MeG, O(6)-MeG, and N(3)-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/11922
關聯:	Anal Bioanal Chem. 2012 Jan;402(3):1199-208.
显示于类别:	[公共衛生學系暨碩士班] 期刊論文

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