The depurination of DNA bases producing an abasic site is a frequent lesion in DNA. This process may occur spontaneously, induced by chemical carcinogens, radiation, oxidative stress, or as an intermediate of the aberrant base excision repair pathway. Several anti-tumor drugs used in chemotherapy also lead to abasic site formation. If not efficiently repaired, the chemically unstable abasic lesions can lead to DNA strand scission, DNA-DNA cross-links and induce mutations. Therefore, detection of abasic sites can provide information about DNA damage due to an array of reactive species. The most well-established method for measuring abasic sites is the use of a biotin-containing aldehyde-reactive probe (ARP) to react with the deoxyribose residue. Labeling with ARP is a semi-quantitative method that requires external standards. In addition, the labeling process is cumbersome and can produce artefactual abasic sites because the derivatization of the abasic site is performed after the isolation of DNA. Our lab previously developed an unambiguous method for quantification of abasic sites following derivatization with O-4-nitrobenzylhydroxylamine and employing liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The detection limit was 3 abasic sites per 107 nucleotides. In this work, we describe a new LC-MS method for labeling abasic sites. We incubated DNA with O-(pyridin-3-ylmethyl)hydroxylamine (PMOA) and used an organocatalyst to accelerate the derivatization reaction. To mitigate artifact formation, we directly treated PMOA with living cells or isolated nuclei and extracted DNA after removing excess labeling reagent. After addition of 13C-labeled internal standard, the DNA was digested with a cocktail of nucleases, followed by solid phase extraction. The level of labeled abasic sites were quantitated by LC-ESI-MS/MS. The labeling method and stability of the derivatized abasic site were established using single- and double-stranded oligonucleotide containing a specific amount of abasic site. The sensitivity of the method is dramatically improved by the use of pyridinyl functional group, with a limit of detection of 50 fg on column.

Sensitive method for quantitation of abasic sites in isolated and cellular DNA by electrospray ionization tandem mass spectrometry

256th National Meeting (2018)

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