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Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2′-deoxycytidine (5-medC) and 2′-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated ( N7-methylguanine ( N7-meG), O-methylguanine ( O-meG), and N3-methyladenine ( N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O-meG and N3-meA were however found to be undetectable in all lung tissue samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Since methylation at the N-7 and O positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N -methylguanine ( N -MeG), O -methylguanine ( O -MeG), and N -methyladenine ( N -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 -MeG, O -MeG, and N -MeA in DNA hydrolysates. With the use of isotope internal standards (N- N -MeG, d - O -MeG, and d - N -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 -MeG, O -MeG, and N -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 -MeG, O -MeG, and N -MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N -MeG, O -MeG, and N -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. [ABSTRACT FROM AUTHOR]