Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung.
Peter W VillaltaJ Bradley HochalterStephen S HechtPublished in: Analytical chemistry (2017)
Benzo[a]pyrene (BaP), an archetypical polycyclic aromatic hydrocarbon, is classified as "carcinogenic to humans" and is ubiquitous in the environment, as evident by the measurable levels of BaP metabolites in virtually all human urine samples examined. BaP carcinogenicity is believed to occur mainly through its covalent modification of DNA, resulting in the formation of BPDE-N2-dG, an adduct formed between deoxyguanosine and a diol epoxide metabolite of BaP, with subsequent mutation of critical growth control genes. In spite of the liquid chromatography-mass spectrometry (LC-MS)-based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (HPLC)-fluorescence detection of BaP-7,8,9,10-tetraols released from human DNA upon acid hydrolysis, BPDE-N2-dG adducts have rarely if ever been observed directly in human samples using LC-MS techniques, even though sophisticated methodologies have been employed which should have had sufficient sensitivity. With this in mind, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace levels of these adducts. These efforts are directly translatable to the development of sensitive detection of other small molecules using trap-based LC-ESI-MS/MS detection. The developed methodology had a limit of detection (LOD) of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct per 1011 nucleotides (1 adduct per 10 human lung cells) using 40 μg of human lung DNA. To our knowledge, this is the most sensitive DNA adduct quantitation method yet reported, exceeding the sensitivity of the 32P-postlabeling assay (∼1 adduct per 1010 nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts per 1011 nucleotides, respectively.
Keyphrases
- tandem mass spectrometry
- liquid chromatography
- high performance liquid chromatography
- mass spectrometry
- ms ms
- ultra high performance liquid chromatography
- simultaneous determination
- circulating tumor
- high resolution
- gas chromatography
- single molecule
- solid phase extraction
- high resolution mass spectrometry
- cell free
- liquid chromatography tandem mass spectrometry
- endothelial cells
- loop mediated isothermal amplification
- sensitive detection
- real time pcr
- label free
- induced pluripotent stem cells
- gold nanoparticles
- high throughput
- healthcare
- nucleic acid
- pluripotent stem cells
- dna methylation
- endoplasmic reticulum stress
- single cell
- transcription factor