Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

Hydroxyl radical protein footprinting is a covalent labelling method that labels solvent accessible side chains amino acids of proteins with hydroxyl radicals. These radicals can be generated by synchrotron radiolysis of water and UV photolysis of hydrogen peroxide. However, the limited availability of synchrotron sources and use of hydrogen peroxide necessitates the need to develop alternative methods. Hydroxyl radical can also be generated by deep ultraviolet (DUV) photolysis of water. Although water weakly absorbs at 200 nm, its absorption increases under DUV laser irradiation due to hydrogen bond disruption. Previous studies have shown that 193 nm laser irradiation of proteins does not cause protein fragmentation, supporting the feasibility of DUV water photolysis for hydroxyl radical protein footprinting. This dissertation focuses on development of new methods for HRPF and photochemical oxidation of lipids using hydroxyl radical combined with mass spectrometry. A DUV laser photolysis was developed for HRPF, and the method was extended to complex biological system such as tissue. In addition, a similar approach was developed for oxidation of lipids. In the first study, a 193 nm excimer laser was used to produce hydroxyl radicals by water photolysis, and proteins were irradiated with single laser pulses in a static photolysis cell. Cytochrome c protein was photochemically oxidized in a photolysis cell, then the oxidized proteins underwent enzymatic digestion followed by LC-MS/MS analysis. The identified sites of oxidation were consistent with those from other protein footprinting studies. To extend HRPF to complex biological systems, proteins in tissue sections were oxidatively modified using hydroxyl radicals generated by photolysis of hydrogen peroxide with a 266 nm laser. The proteins were then extracted and analyzed by LC-MS/MS. A method was also developed for photochemical oxidation of lipids using hydroxyl radicals generated by UV photolysis of hydrogen peroxide. Oxidation of a phosphatidylcholine standard resulted in 50 % yield with multiple oxidized products, and application to tissue samples showed oxidation of several phosphatidylcholine species. This method offers a promising strategy for double-bond localization in unsaturated lipids when coupled with LC-MS/MS analysis.

Date

3-12-2026

Committee Chair

Murray Kermit

LSU Acknowledgement

1

LSU Accessibility Acknowledgment

1

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