DNA adducts constantly arise from endogenous metabolism and environmental exposure. This usually results in an array of DNA lesions. For example, exposure to cigarette smoke is known to give rise to a plethora of alkylated DNA lesions. Therefore, a DNA adductomic approach is required to robustly quantify DNA adducts in cellular and tissue DNA. Recent developments in scheduled selected-reaction monitoring (SRM) analysis of peptides and proteins have afforded highly sensitive and high-throughput analytical methods for samples in complicated matrices. In this respect, normalized retention time (iRT) has been widely used for scheduled SRM analyses of peptides, where iRT is empirically derived from the retention time of analyte of interest relative to that of the standard peptides. By using a similar method, we established the iRT values of approximately 20 synthetic modified nucleoside standards from the retention times of canonical 2′-deoxynucleosides and ribonucleosides on an nLC-nESI-MS/MS/MS system (LTQ XL) as well as on a high-resolution nLC-nESI-MS/MS system (Q Exactive Plus). Since some DNA adducts are bulky and may not be released quantitatively during the enzymatic digestion process, release efficiencies of DNA adducts were evaluated by digesting calf thymus DNA spiked with different amount of adduct containing oligodeoxynucleotides (ODNs). We examined the release efficiencies of several DNA adducts, including the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyadenosine (S-cdA) and 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that the release of S-cdG from a lesion-containing oligodeoxynucleotide was nearly quantitative, whereas only 15% S-cdA could be released as a mononucleoside under the same digestion conditions. We are now in the process of employing this DNA adductomic approach for assessing the formation and repair of oxidatively induced and alkylated DNA lesions.

Scheduled LC-SRM method for targeted DNA adductome analysis

256th National Meeting (2018)

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