Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

Abstract

Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. DNA adducts reflect the internal exposure to genotoxicants and can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MSn) is the most common method for biomonitoring DNA adducts, generally targeting single exposures and measuring up to several adducts. However, the data often provide limited evidence for a role of a chemical in the etiology of cancer. An “untargeted” method is required that captures global exposures to chemicals, by simultaneously detecting their DNA adducts in the genome; some of which may induce cancer-causing mutations. We established a wide selected ion monitoring tandem mass spectrometry (Wide-SIM/MS2) screening method utilizing ultra-performance-LC nanoelectrospray ionization Orbitrap MSn with on-line trapping to enrich bulky, non-polar adducts. Wide-SIM scan events are followed by MS2 scans to screen for modified nucleosides by co-eluting peaks containing precursor and fragment ions differing by -116.0473 Da, attributed to the neutral loss of deoxyribose. Wide-SIM/MS2 was shown to be superior in sensitivity, specificity, and breadth of adduct coverage to other tested adductomic methods with detection possible at adduct levels as low as 4 per 109 nucleotides. Wide-SIM/MS2 data can be analyzed in a “targeted” fashion by generation of extracted ion chromatograms or in an “untargeted” fashion where a chromatographic peak-picking algorithm can be used to detect putative DNA adducts. Wide-SIM/MS2 successfully detected DNA adducts, derived from chemicals in the diet and traditional medicines and from lipid peroxidation products, in human prostate and renal specimens.

The Future of DNA Adductomic Analysis

“…Ideally, direct comparisons of various DNA adductomic methodologies using identical samples would provide a true measure of their relative analytical power. Turesky and coworkers have recently performed a comparison of targeted DNA adductomics methodologies of Orbitrap-based DIA-WideSIM/MS 2 and DDA-CNL/MS 3 analyses and triple quadrupole-based CNL and pseudo-CNL analyses, all four of which were performed on the same samples with identical chromatography and ion source conditions [ 57 ]. Levels of synthetic DNA adduct standards were spiked in calf thymus DNA and analyzed with the different methods.…”

DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans

“…Recently, our laboratory adapted the sequential window acquisition of all theoretical fragment‐ion spectra (SWATH) approach, pioneered for proteomic investigations, to develop a DIA method, termed wide‐SIM/MS 2 , to screen for multiple DNA adducts using hybrid Orbitrap‐Fusion MS . Multiple targeted‐SIM scans with 30 m/z width were performed, and each scan was followed by an HCD‐MS 2 to fragment all ions detected in the previous SIM scan.…”

“…Moreover, data files obtained by wide‐SIM/MS 2 can be retrospectively analyzed to probe for newly found or unexpected adducts in previously assayed samples. However, a limitation of the wide‐SIM/MS 2 is that fragmentation data at the MS 2 scan stage principally provides only one product ion [M + H − dR] + , and another analysis using targeted MS 3 is required to confirm the identity of the DNA adduct . In this context, MS E with a Q‐TOF, or Orbitrap MS instrument may be advantageous to screen for DNA adducts, if the collision energy is sufficient to further fragment the aglycone ion.…”

“…Some DDA and DIA scanning methods developed for proteomics have migrated to the field of DNA adductomics and have been successfully used to scan for DNA adducts . Another factor critical for the success of DNA adductomics is the availability or the development of accompanying informatic tools.…”

“…Unlike the fields of proteomics or metabolomics, which have robust tools for data analysis, workflows for the automated analysis of DNA adductomics data, including common neutral loss and MS 3 spectra acquisition generated by DIA or DDA HRAMS ion trap based methods, have yet to be established. A mass spectral database on the DNA adducts also must be established for use by the DNA adductomic community . With the advancements of these technologies, DNA adductomics will take its place next the established—omics methodologies.…”