Collision-induced dissociation studies of protonated ions of alkylated thymidine and 2'-deoxyguanosine.

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O-4-alkylthymidine (O-4-alkyldT), O-2-alkyldT, O-6-alkyl-2'-deoxyguanosine (O-6-alkyldG), and N-2-alkyldG through MS3 analysis. The MS3 of O-2- and O-4-MedT exhibit different fragmentation patterns from each other and from other O-2- and O-4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O-2-alkyldT, O-4-alkyldT, and O-6-alkyldG adducts carrying larger alkyl groups, whereas O-6-MedG mainly undergoes elimination of ammonia. The breakdown of N-2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.