Protein oxidation can cause aggregation, fragmentation, and affect enzymatic activity and binding partner interactions. Protein oxidation is implicated in a range of agerelated pathologies including neurodegeneration and cancer. The VHR and PTEN phosphatases studied are sensitive to oxidation and regulated by protein-protein interactions. PTEN acts by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, negatively regulating the Akt pathway as part of a signalling control network that can protect against apoptosis, and is involved in the regulation of cell fate regulation and cancer. VHR is involved in neural development and cancer. A technology workflow for detecting protein oxidation and to correlate oxidative modifications to enzymatic activity and protein-protein interaction was developed; which may contribute towards the advancement of fundamental science as well as potential therapeutic and biomarker target identification in proteins. The technology platform consists of the mass spectrometric technique MS2 to detect, validate, map and quantify oxidative modifications. The technology workflow consists of enzymatic activity assays to correlate modification with changes in activity, targeted MS2 and statistical analysis. The fundamental and distinct contribution to knowledge in this thesis is a systematic mapping of protein oxidative modifications over a range of oxidants and concentrations of hypochlorous acid (HOCl), 3-morpholino-sydnonimine (sin-1) and tetranitromethane for VHR (vaccinia H1 related) and PTEN (phosphatase and tensin homolog on chromosome 10), including modification identification including active site residues and putative binding domain, mapping the relative abundances of those modification and statistically correlating them to changes in enzymatic activity. Additional contributions to knowledge have been i) the nonspecificity and complexity of oxidation profiles and oxidant damage of nitrating agents, that have largely been proposed to be specific without substantial oxidative capacity and ii) expanding the known interactome of VHR (vaccinia H1 related) through array and co-immunoprecipitation.
- mass spectrometry