Oxidative stress, caused by excessive reactive oxygen species (ROS), is evident in many chronic diseases. However, there is limited mechanistic information on how oxidation of multiple proteins is causing cell and tissue dysfunction. A systems biology approach has the potential to provide novel insights into mechanisms of action.
Protein thiol oxidation is the focus of this research because thiol groups (-SH) on cysteine residues of proteins are particularly sensitive to oxidation. As a consequence, changes in the function of multiple proteins caused by thiol oxidation affect many aspects of cell and tissue function. In preliminary work using gel electrophoresis and mass spectrometry identification, multiple proteins were observed to undergo thiol oxidation in muscles of a dog model for dystrophy.
In order to expand the number of proteins that can be identified in complex biological samples, high throughput proteomic techniques using gel-free mass spectrometry will be valuable. In this research, proteins will be tagged with two differential mass tags and a data independent acquisition method – SWATH – will be applied on the 5600 TripleTOF (ABSciex) mass spectrometer platform in order to identify the thiol oxidised proteins in tissue samples. Information about the sites of oxidation on proteins and the degree of oxidation will also be generated. Further investigation into protein locations, and involvement in molecular networks may elucidate molecular pathways that contribute to cellular dysfunction in diseases where oxidative stress is evident (e.g. muscular dystrophy).