Quantitation of 7-Ethylguanine in Leukocyte DNA from Smokers and Nonsmokers by Liquid Chromatography–Nanoelectrospray-High Resolution Tandem Mass Spectrometry

Abstract

There is considerable evidence for exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [15N5]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H]+ → m/z 152.05669 [Gua + H]+ for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (± S.D.) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6 – 181) fmol/μmol Gua while that from nonsmokers was 41.3 ± 34.9 (range 9.64 – 157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.

NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

“…To achieve detection limits in the low fmol to amol range (1 per 10 8 –1 per 10 9 nucleotides) as required for our in vivo studies, we employed nanoLC/ESI + -HRMS 3 methodology on an Orbitrap Velos mass spectrometer. We and others have recently reported the use of high resolution mass spectrometry for DNA adduct analysis in complex samples and have shown that it dramatically reduces the matrix background, leading to greatly improved signal to noise ratios [37, 45, 46]. …”

“…DNA from human blood samples was isolated using the manufacturer’s protocol for DNA purification from buffy coat (Qiagen, Valencia, CA) [37] with minor modifications [36]. DNA isolation from rat liver was performed using the manufacturer’s protocol for DNA purification from tissue (Qiagen, Valencia, CA).…”

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung

“…29 Using that study as a starting point, we increased the sensitivity of the analysis for BPDE- N 2 -dG by 4 to 5 orders of magnitude, allowing the detection of BPDE- N 2 -dG at levels as low as 1 adduct per 10 11 nucleotides (S/N = 2) vs the previously described detection limit of 53 adducts per 10 8 nucleotides (S/N = 6). 29 Some of this improvement in sensitivity can be accounted for by the larger amount of DNA analyzed (~40 μg vs 2 μg), however the majority of the improvement is attributable to several important features which optimized our existing HRAM MS 2 nanospray quantitation methodology 23–26 as discussed below, and can potentially be used to improve the sensitivity of other quantitative assays.…”

Occurrence, Biological Consequences, and Human Health Relevance of Oxidative Stress-Induced DNA Damage

“…88,159,160,163,165 Recently, high-resolution Orbitrap mass spectrometers have been used for the detection of alkylated DNA lesions induced by carcinogens present in tobacco smoke. 344,345 Owing to their accurate mass measurement capability and high sensitivity, we expect more applications of this type of instruments for the quantification of low levels of oxidatively generated DNA lesions in cellular and tissue samples in the future. The improvement in LC-MS-based quantification techniques will also enable systematic adductomics research, 346–348 which may facilitate the discovery of novel oxidatively induced DNA lesions involved in the pathogenesis of human diseases.…”