Increasing life expectancy combined with an unbalanced diet in Western societies promotes the development of diseases such as obesity, type 2 diabetes mellitus and non-alcoholic fatty liver disease. One potential molecular cause for the progression of age- and diet-related diseases is an imbalance in redox homeostasis favouring oxidative processes. Understanding the redox proteome is crucial for the selective modulation of the redox sensor system for therapeutic approaches. This study systematically analysed the impact of diet and aging on the redox proteome of the liver as a central metabolic organ. Therefore, 84 Wistar ats (Rattus norvegicus) of both sexes were fed a moderate Western diet (WD) mimicking dietary patterns in central Europe (WD, 36 kcal % fat, 13 % sugar) or a standard diet (SD, 10 kcal % fat) for up to 78 weeks. Various in vivo tests (glucose tolerance, activity, blood pressure) were conducted, and liver tissues were collected at defined time points (26, 52 and 78 weeks of age) for further analysis.

Changes in cysteine redox status may reflect alterations in redox signalling pathways. In this study, non-oxidized cysteine peptides were labelled using N-ethylmaleimide (NEM), providing an indicator of cysteine oxidation status. A decrease in relative protein levels of NEM-sites indicates an increase in the oxidized status of the proteins. Using sequential alkylation and modified C-STrap sample preparation, the samples were fractionated after tryptic digestion into a non-cysteine fraction containing NEM-labelled non-oxidized cysteine peptides as well as cysteine-free peptides and a N-propylmaleimide (NPM)-labelled oxidised cysteine fraction. Following TMT-labelling the samples were analysed using high-resolution LC-MS/MS (Orbitrap). Analysis of NEM-sites revealed 84 and 78 significantly regulated redox-sites for male and female rats, respectively, differing between WD and SD. In females, NEM-sites involved in pyruvate and lipid metabolism were enriched during pathway analysis. In males, pathway enrichment highlighted proteins involved in purine and pyrimidine metabolism and energy homeostasis due to altered redox profiles as a consequence of WD. Age-related analysis of NEM-sites showed a higher number of regulated proteins in males, with 16 sites compared to 7 in females. For instance, males exhibited among other observations a reduction in NEM-sites of glutathione S-transferases and increased levels of thioredoxin (TXN), suggesting increased oxidation levels with age and potential changes in redox signalling pathways, respectively. TXN's role in regulating transcription factors and protecting against oxidative stress may be affected by changes in its oxidation status. These findings provide insights into early metabolic changes and adaptation mechanisms in rats under a moderate WD, with a notable sex-specific liver proteome profile that may explain differences in prevalence and manifestation of diet- and age-related pathologies.