title: Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction.
abstract: 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.
authors.affiliation: Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan.
authors.forename: Chiung-Wen
authors.initials: CW
authors.lastname: Hu
authors.forename: Chih-Ming
authors.initials: CM
authors.lastname: Chen
authors.forename: Hsin Hui
authors.initials: HH
authors.lastname: Ho
authors.forename: Mu-Rong
authors.initials: MR
authors.lastname: Chao
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D019175:DNA Methylation; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D007201:Indicator Dilution Techniques; D007554:Isotopes / Q000032:analysis; D011687:Purines / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000191:economics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*
chemical_list: D007554:Isotopes; D011687:Purines; D006147:Guanine; D004247:DNA; C427203:calf thymus DNA; C008449:O-(6)-methylguanine