Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry

Abstract

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

Mass Spectrometric Mapping of the DNA Adductome as a Means to Study Genotoxin Exposure, Metabolism, and Effect

“…[12][13][14] More than a decade ago Koc et al stated that the only disadvantage of MS in the field of DNA adduct analysis, was its sensitivity. 28 Over the years, sensitivity has continued to improve 6,14 as different research groups have focused on optimization of DNA adduct detection methods with MS [30][31][32][33][34][35][36][37] , including research into non-manual data mining and sequencing to locate DNA adduction sites. 38,39 Due to ongoing technical advancements and the use of stable isotope labeled internal standards, MS currently offers a reliable tool to measure low DNA adduct levels with the highest specificity.…”

“…31 The aforementioned research demonstrates that tandem MS can reveal hundreds of putative DNA adducts in DNA samples and thus holds great potential for biomarker discovery. However, one disadvantage of tandem MS/MS is the loss of sensitivity with CNL or the need for SRM transition optimization for each different DNA adduct in order to achieve sufficient detection sensitivity with pseudo-CNL.…”

“…However, one disadvantage of tandem MS/MS is the loss of sensitivity with CNL or the need for SRM transition optimization for each different DNA adduct in order to achieve sufficient detection sensitivity with pseudo-CNL. 27,31 Secondly, since triple quadrupoles only allow low resolution data acquisition, and DNA adduct databases providing MS/MS spectra are not available, DNA adduct identity confirmation is dependent on the availability of analyte standards or the use of additional analytical techniques.…”

“…Within this framework, the use of capillary or nano capillary LC coupled to micro-or nano-ESI-MS could provide a rise in sensitivity. 14,31 Due to the lower sample flow rates of capillary LC, the ionization and ion sampling efficiency in the electrospray source increase significantly, resulting in a higher amount of ions in the MS system, an improved sensitivity and low mass detection limits. 14 An additional technique that could be used to achieve an increase in sensitivity, consists of miniaturized separation techniques like LC-chip.…”

A nontargeted screening method for covalent DNA adducts and DNA modification selectivity using liquid chromatography-tandem mass spectrometry

“…In this way, the structurally modified and nonmodified nucleosides can be quickly screened in the chromatogram. For example, this technique was used to screen polycyclic aromatic hydrocarbons (PAHs) related DNA adducts by incorporating with selected reaction monitoring (SRM) [16]. The SRM is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection.…”