Conclusions
CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.
Methods
A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS2 or a matrix-assisted laser desorption/ionization (MALDI)-MS technique.
Results
Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 109 nucleotides (S/N ~30). Conclusions: CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.