BPS2026 – Investigation of residue-specific radiation damage of peptides under different radiation doses, dose rates, and oxygen availability

X-ray footprinting mass spectroscopy (XFMS) is a method of structural biology, wherein the pattern of oxidative damage to a protein in solution is used to determine its structure and function. The irradiation of water produces hydroxyl radicals, which oxidatively and covalently modify a protein at solvent-accessible positions. Proteins are digested and then undergo liquid chromatography mass spectroscopy (LC-MS) to identify site-specific oxidation. This information can be used to assess protein structure and dynamics, but here we have used the method to further investigate the differences in modification of short peptides based on radiation dose rate, source, and oxygen availability in solution. Eight peptides with a single residue change differentiating them were synthesized (EDLAXLK, where the variable X position is F, G, H, I, M, R, P, or Y). Anaerobic and aerobic samples diluted in phosphate buffer were irradiated using the advanced light source (ALS) synchrotron at Lawrence Berkeley National Laboratory (LBNL) (134.6 Gy/s), the Berkeley Lab Laser Accelerator (BELLA) Center’s laser-driven proton beamline (10 7 Gy/s) and a low-dose-rate XRAD320 X-ray machine (0.020 Gy/s) at various doses. Oxidative modifications, specifically +16 Da, +32 Da, and +14 Da were quantified by LC-MS/MS post-irradiation. Preliminary results generally show less modification under anaerobic conditions, but under those conditions, peptides irradiated at the ALS exhibited more modification than XRAD counterparts, with some interesting exceptions. Additionally, the oxygen availability in solution has a larger impact on modification with XRAD irradiation as compared to ALS irradiation. We hope to further characterize the oxidative modification pathways which follow irradiation of peptides and proteins to improve the XFMS method, as well as better understand the roles of oxygen and radiation dose rate in the damage to proteins during radiotherapy cancer treatments.