Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (BIO2015-67580-P, PGC2018-097019-B-I00 and RTI2018-093864-B-100),the Ministry of Economy, Industry and Competitiveness (BFU2016-80006-P) through the Carlos III Institute of Health-Fondo de InvestigaciónSanitaria grant PRB3 (IPT17/0019 -ISCIII-SGEFI / ERDF, ProteoRed), the Fundació MaratóTV3 (grant 122/C/2015) and “la Caixa” Banking Foundation (project code HR17-00247). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)S