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.

DNA Crosslinkomics: A Tool for the Comprehensive Assessment of Interstrand Crosslinks Using High Resolution Mass Spectrometry

“…Alternatively, to avoid potential release bias of the hydrolysis enzymes, Strategy II can also be applied to DNA samples following acid and thermal hydrolysis, in which all the DNA crosslinks will be released as nucleobase crosslinks. An example of the use of Strategy II to measure the DNA crosslinks in the CLB-treated DNA following acid-thermal hydrolysis 25 is provided (Figure S10). Using Strategy II, the amounts of DNA crosslinks following acid-thermal hydrolysis were ~2–20-fold more than those following enzymatic hydrolysis.…”

“…After incubation with formaldehyde or CLB, DNA was precipitated and recovered using a solution of NaI, 2-propanol, and ethanol, as previously described. 25 The DNA pellet obtained was dissolved in 500 μ L of 0.1 mM DFO solution, to a DNA concentration of ~500 μ g/mL. The DNA recovery was nearly 100%, as determined by the dG content using LC-MS/MS.…”

Genomewide Dam Methylation in Escherichia coli during Long-Term Stationary Phase

“…DNA was hydrolyzed to nucleobases using a protocol modified from references 51 and 52. Triplicates of 10 µg of dried genomic DNA was dissolved in 100 µl of 98% LC-MS grade formic acid (FA) (Fisher Scientific, Waltham, MA) sealed in glass tubes and hydrolyzed in a covered heat block at 90°C for 6 h. Hydrolysis was monitored by LC-MS as described below, and loss of genomic DNA was observed visually on a 0.7% agarose gel.…”

Recent Advances in The Bioanalysis of Modified Nucleotides in Epigenetic Studies

“…In 2010, HPLC coupled to a sensitive MS detector was used in the identification of 5-hmC in mouse brain tissues [58]. 2 years later, LC-MS/MS was used for the determination of 5-mC and 5-methyl-2´deoxycytidine in urine samples [59] and methylated DNA lesions, such as N(7)-methylguanine, O(6)-methylguanine and N(3)-methyladenine [60]. Moreover, isotope LC-ESI-MS and immunohistochemistry was used to study the distribution of 5-hmC in various mouse tissues and cell types, and to detect 5-fC, 5-caC and 5-hydroxymethyluracil [61].…”