Ultraviolet (UV) exposure induces cross-linked pyrimidine dimers in nucleic acids, primarily forming cyclobutane pyrimidine dimers and 6–4 pyrimidine–pyrimidone adducts. These photoproducts exist in multiple isomeric forms, and various dimeric combinations involving thymine, cytosine, and uracil have been documented since the 1960s. Mass spectrometry (MS) has been pivotal in identifying these species, although condensed-phase spectroscopy remains essential for full structural elucidation. This study integrates MS with gas-phase infrared (IR) spectroscopy to obtain vibrational spectra (800–1900 cm–1) of UV-induced photoproducts from mono- and dinucleotides. Following nanoelectrospray ionization and in-source collision-induced dissociation, fragment ions─commonly used in tandem MS experiments to identify the photoproducts─are embedded in superfluid helium clusters at 0.37 K to measure high-resolution IR action spectra. These spectra are then compared with density functional theory-calculated spectra of various candidate isomers to facilitate structural assignment without reference standards. This combined approach enables detailed characterization of complex, low-abundance biomolecules beyond the reach of conventional MS.
