Developing chemical mass spectrometry probes to assess the production of reactive oxygen species in vivo

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Within our bodies, and the bodies of all animals, the oxygen we breathe undergoes a side reaction which leads to the production of small molecules called 'reactive oxygen species' (ROS). These ROS are responsible for the oxidative damage associated with ageing. Indeed, it has been argued that they cause the ageing process itself and the conditions, such as nuerodegeneration, associated with it. ROS are also used in important signaling pathways within living organisms. However, while there is a lot of circumstantial evidence pointing to the importance of ROS within living creatures, there is also a considerable amount of uncertainty about the extent of their role. This is because ROS are very short lived and difficult to measure, so while they can be quantified in the test tube, it is currently not possible to work out how much ROS are present inside an organism. Because of this difficulty, we are often uncertain about how important ROS are in a range of important biological processes, such as ageing, disease, growth and cancer. To overcome this difficulty we will develop a new method using molecular probes that are designed to go to mitochondria, the parts of the cells that make up our bodies which are believed to be the main sources of ROS. The targeting to mitochondria relies on a simple physicochemical effect whereby lipophilic cations capable of crossing membranes accumulate in the mitochondrial matrix. Once there, the probe molecules will react with the ROS to form distinctive products. The ratio of the starting molecule to its product will then give an indication of the amount of ROS in isolated mitochondria and in the mitochondria of whole cells and animals. Similar probes are designed to remain outside cells so that ROS can be detected in the extracellular environment, particularly in the circulation. Our approach has to be very sensitive in order to assess the small changes that occur within organisms. To facilitate this, the probes are designed so that they can be detected in complex biological mixtures selectively and sensitively, even when present at very small amounts, by a technique called liquid chromatography-tandem mass spectrometry. Through the new probes' combination of targeting to particular locations in the cell or extracellular environment along with their built in feature that allows very sensitive detection by mass spectrometry, we will be able measure ROS in experimental animals and thereby work out for the first time the detailed contributions of ROS to many important biological processes, most significantly to the process of ageing itself. Antioxidants targeted to remain outside cells will also be developed as an intervention that will allow us to assess how the specific contribution of oxidative stress in the circulation and extracellular matrix affects the process of ageing as a whole.