Structure Elucidation of DNA Interstrand Cross-Link by a Combination of Nuclease P1 Digestion with Mass Spectrometry

DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand cross-link. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5ʹ,8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs.