OP37 - Redox proteomic approaches to understanding age-related changes in redox signalling

Abstract

Redox modifications of regulatory amino acids offers a dynamic and versatile means to rapidly alter the activity or structure of proteins in response to external and internal stimuli. These stimuli often refer to endogenously generated reactive oxygen or reactive nitrogen species (ROS/RNS), which play a role in normal cellular functioning including glycolysis, oxidative phosphorylation, biogenesis, autophagy, transcription, translation. The activities of redox sensitive proteins are often determined by the redox state of specific cysteine (Cys) residues located in catalytic sites or involved in co-factor binding. Disruption or dysregulation of many of these pathways by excessive or diminished levels of ROS/RNS signalling has been suggested to play a crucial role in ageing and many metabolic diseases. Skeletal muscle provides an ideal model to study age related changes in redox signalling, as muscle generates ROS/RNS during contractions and ageing is associated with a loss of skeletal muscle mass and function, with particular muscle types more susceptible than others. In this presentation, I will describe techniques that we have applied to study the effects of age on the redox proteome of a number of different skeletal muscles (Soleus, Gastrocnemius, and Vastus lateralis). Combining global label free proteomics and differential Cys labelling allowed the relative quantification of the redox state of specific Cys residues in the context of the protein’s abundance. Once redox sensitive Cys residues have been identified a targeted redox proteomic approach using parallel reaction monitoring, can accurately quantify the reversible oxidative state of individual Cys residues. Results indicate that muscles from old mice have a reduced redox flexibility and that muscle types respond differently to the effects of ageing. Differences we observed in the redox proteome are a reflection of the age related changes in redox signalling within muscle types.