Protein aggregation capture assisted profiling of the thiol redox proteome

Abstract

Oxidative damage is critical in various diseases, including cardiovascular and neurological conditions. Thiol redox reactions, acting as oxidative stress sensors, influence protein structure and function. Redox proteomics based on differential alkylation of reduced and oxidized Cys forms using mass spectrometry enables comprehensive analysis of thiol redox status in cells and tissues. We introduce PACREDOX, an innovative redox proteomics approach based on the Protein Aggregation Capture (PAC) protocol and we demonstrate its compatibility with library free data-independent acquisition (DIA). PACREDOX reduces preparation time and costs compared to traditional methods, such as FASILOX, while maintaining thiol and proteome coverage. To enable library-free DIA, we corrected in silico spectral libraries in DIA-NN using experimental retention time data from beta-methylthiol-modified peptides. PACREDOX with DIA quantified 4,000 protein groups and [~]45,000 modified peptides in myocardial tissue from a porcine model of atrial fibrillation, including over 8,000 cysteine-containing peptides, 30% of which were reversibly oxidized. Benchmarking PACREDOX and DIA against FASILOX in a myocardial infarction model reflects the potential and efficiency of this methodology to study oxidative damage. Overall, PACREDOX offers a high-throughput, cost-effective strategy for thiol redox proteome analysis, compatible with label-free quantitative workflows.