Abstract 4791: Development of a high resolution mass spectrometry method for the observation and characterization of unknown adducts in human DNA

Abstract

Abstract The development of analytical approaches for identification of DNA adducts may provide important tools to help clarify their role as biomarkers of exposure and as potential markers of cancer risk. New sensitive mass spectrometric methods are needed to screen for previously unidentified modified bases to allow for identification of adducts of multiple types, potentially leading to the identification of base modification patterns in DNA from human tissues. Liquid chromatography-mass spectrometry (LC-MS) is now the method of choice for characterization and quantitation of DNA adducts in tissue samples. We have developed a unique LC-High Resolution-MSn-based DNA adductomics approach which has the potential to identify previously uncharacterized DNA adducts in human tissues because it possesses the required combination of sensitivity, specificity, and generation of chemical information that is absent from previous MS techniques. DNA hydrolysis produces deoxynucleosides (dR) which upon ionization can undergo fragmentation resulting in loss of deoxyribose, resulting in an overall [M+H - 116.0474]+ ion. This universally observed fragmentation can be used to identify the analyte as a dR. The methodology described here uses the appearance of this fragment ion at high mass tolerance to trigger a second fragmentation step (MS3) leading to production of fragment ion spectra which provide additional structural information. An additional fragmentation step (MS4) is triggered for those MS3 spectra, which contain ions corresponding to one of the four nucleobases to provide confirmation of base identity. The final output is an LC chromatogram of DNA adducts in which each peak provides an accurate mass sufficient for unambiguous molecular formula determination and extensive fragmentation data potentially leading to its structural identification. We performed preliminary experiments to test the performance of our approach on mixtures of 18 DNA adducts selected to represent modifications of all four nucleobases and modifications at various reactive sites and with differing polarity. All 18 DNA adducts showed clear, sharp and symmetric peaks in the full scan and [M+H - 116.0474]+ ions upon MS2 fragmentation which then triggered the MS3 event on the product ions. The fragmentation spectrum of each adduct contained the ion corresponding to the mass of the base it was derived from and triggered the fragmentation of the base ion in the MS4 event. To test the effect of a real sample matrix on DNA adduct detection, the mixture of 18 DNA adducts was added to a human leukocyte DNA sample which had previously been passed through the purification and enrichment steps used for our LC-MS/MS analysis of N2-ethyl-dGuo. All 18 DNA adduct standards were detected as clear and sharp peaks. These results clearly demonstrate the potential of this method for DNA adduct detection and identification in human tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4791. doi:1538-7445.AM2012-4791