Highly reactive free radicals are generated by exposure of cellular material to ionizing radiation and may produce DNA-protein cross-links (DPC) among other types of damage which, if unrepaired, may play a significant role in mutagenesis, carcinogenesis, aging and cell death. Present structural information on DPC, relying on mass spectrometric identification of derivatized amino acids-nucleobase dimers, will be extended to intact macromolecules using electrospray ionization mass spectrometry (ESI-MS). Studies will be conducted with irradiated components from a defined nucleosome model and also those from isolated cellular nucleosomes. Cross-linked histone-DNA and peptide-nucleotide sample preparation will involve biochemical and molecular techniques for isolation, followed by ESI-MS, providing specific qualitative information on covalent linkage for DPC. The multiple charging observed in ESI will enhance the collision induced dissociation for multiple sequential tandem mass spectrometric characterization and sequencing of enzymatically derived peptide-nucleotide species. These studies will be extended by the development of an ESI source interfaced to an ion trap mass spectrometer (ITMS). The long duty cycle and ion storage in ITMS will maximize sensitivity and permit unique extended tandem mass spectrometry (MSn) protein sequencing and qualitative studies. Biochemical and mass spectrometric methods will locate DPC specifically within nucleohistone amino acid and DNA base sequences.
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