On the utility of immobilized phenylarsine oxide in the study of redox sensitive cardiac proteins.

Reactive protein cysteine thiols are critical to sensing and transducing oxidant signals, often by induction of disulfide bonds that alter their activity or interactions. Identifying such redox active proteins nowadays is mostly achieved using thiol redox proteomics with such datasets increasingly available. Subsequently, we are challenged with determining how changes in the redox state of a protein of interest alters its activity or interactions and how this affects physiology or disease progression including in vivo scenarios. Such studies necessitate the measurement of how the protein redox state changes with health or disease-related interventions, with it not always being practicable to resort back to resource-intensive proteomics to achieve this. In some proteins, oxidation to a disulfide state causes a non-reducing gel-shift, but this is mostly not the case and so other efficient approaches are required to index changes in redox state. Here we assessed the utility of immobilized, solid-phase phenylarsine oxide (PAO-Sepharose) as a tool for indexing the thiol redox state of candidate proteins in cardiac samples from in vivo interventions associated with oxidative stress. PAO-Sepharose, which binds proteins with proximal reduced thiol pairs but not when they form a disulfide, was also used to identify proteins that that are oxidised in isolated perfused mouse hearts exposed to hydrogen peroxide or diamide using proteomics. This together with complementary studies using a cardiac-specific FLAG-Thioredoxin-1C35S-HA transgenic 'trap-mutant' mouse model allowed identification of heart proteins susceptible to oxidant-induced disulfide bond formation using proteomics. Thus, two in vitro approaches identified putative cardiac thiol redox sensor proteins that were then assessed with in vivo follow-up studies for their susceptibility to oxidation during endotoxemia induced by lipopolysaccharide or type I diabetes induced by streptozotocin in mice. Of five proteins selected for further analysis by PAO-Sepharose binding, two, namely apoptotic protease activating factor 1 interacting protein (APIP) and γ-glutamylcyclotransferase (GGCT), displayed significantly lower affinity capture from hearts from lipopolysaccharide- or streptozotocin-treated mice, consistent with oxidation of their vicinal thiols. We conclude that PAO-Sepharose is an effective and accessible tool for identifying oxidant-sensitive protein thiols in both ex vivo and in vivo models of oxidative stress. As increasing numbers of thiol redox proteins are identified, PAO-Sepharose binding is an efficient method to determine if they change their oxidation state during interventions relevant to health and disease.