The study of radiation induced DNA-protein crosslinks by electrospray ionization mass spectrometry

The authors have used peptide-thymine and histone-thymine solutions to model protein-DNA cross-linking chemistry induced in intact chromatin by low dosage of g-irradiation. Induced thymine crosslinking to model peptide systems has been evaluated by on-line liquid chromatography-electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) with sensitivity comparable or superior to conventional GC-MS determinations. Radiation damage at doses as low as 0.1 Gy can be detected by this method. Additionally, thymine modified H2B can also be examined by ESI-MS and tandem-MS of the intact protein and proteinase digests. Limited information on the sites of thymine crosslinking can be obtained by tandem mass spectrometry on the intact multiply charged molecular species. More detailed information on the sites of thymine-protein crosslinking is obtained by on-line LC-ESI-MS of selective proteolysis products of the modified histones. Further MS-MS experiments on the selective proteolysis products will reveal specific modified amino acids and their sequence location. These methods reveal the nature, extent and site of radiation induced modification of the oligopeptides. Studies are being extended to the examination of the radiation induced covalent interactions between histones and oligonucleotides in higher states of organization. The eventual object is to study DNA-protein crosslinking interactions in model and native genomic nucleosome systems