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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM047423-01
Application #
3306918
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1992-08-01
Project End
1996-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Battelle Pacific Northwest Laboratories
Department
Type
DUNS #
032987476
City
Richland
State
WA
Country
United States
Zip Code
99352
Teske, J G; Edmonds, C G; Deckert, G et al. (1997) Structural features of Glycera dibranchiata monomer hemoglobins. Primary sequences of monomer hemoglobin components II and III. J Protein Chem 16:139-50
Lipton, M S; Fuciarelli, A L; Springer, D L et al. (1997) Analysis of radiation induced nucleobase-peptide crosslinks by electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 11:1673-6
Grammer, J C; Loo, J A; Edmonds, C G et al. (1996) Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin. Biochemistry 35:15582-92
Weir Lipton, M S; Fuciarelli, A F; Springer, D L et al. (1996) Characterization of radiation-induced thymine-tyrosine crosslinks by electrospray ionization mass spectrometry. Radiat Res 145:681-6
Siegel, M M; Huang, J; Lin, B et al. (1994) Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry. Biol Mass Spectrom 23:186-204