Ion Mobility Separation of Six Isomeric Cyclobutane Thymidine Photodimers Relevant to the Photochemistry of Non-B DNA Structures

Thymidine dimers, which exist in multiple stereoisomeric forms, are important UV-induced DNA lesions, yet their structural differentiation remains challenging. In this study, we leveraged trapped ion mobility spectrometry coupled with time-of-flight mass spectrometry (timsTOF Pro) to resolve all six possible isomeric thymidine dimers. Building on preliminary analysis using traveling wave ion mobility, we optimized TIMS scan parameters to achieve baseline resolution of all six isomers. By integrating experimental ion mobility data with density functional theory (DFT)-based structural modeling and trajectory-method CCS predictions using IMoS software, we demonstrated that syn-isomers exhibit conformations more compact than those of anti-isomers. This work establishes a robust platform for gas-phase structural resolution of DNA photoproducts and may complement isotopic dilution-based strategies for quantification of these important photoproducts in vitro and in vivo. The combination of experimental and theoretical ion mobility provides new insights into the stereochemical diversity and structural dynamics of not only thymidine photo dimers but also dinucleotide photoproducts.