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

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.

The measurement of Reactive Oxygen Species (ROS) levels in vivo is a major unmet need in biology. Current methods for ROS assessment are done by the use of fluorescent probes, or less frequently by chemiluminescence or electron paramagnetic resonance. While these methods work for cells in culture, they cannot generally be applied to whole organisms. Consequently, while there is considerable circumstantial evidence for a significant contribution of ROS to a wide range of biological processes, such as ageing, pathology, development, hypoxia-sensing and malignancy, the details are sketchy. ROS formation leads to the accumulation of oxidatively damaged biomolecules, however inferences on the roles of ROS in vivo based on the extent of accumulation of oxidative damage markers are flawed because the extent of accumulation is a combination of the rates of damage, repair and turnover, all of which vary. Therefore new methods are urgently required to assess ROS in vivo. To do this we have designed a range of mass spectrometry probes that will enable us to selectively and sensitively detect different types of ROS in vivo. We will create probe molecules that contain a targeting group to direct them to either mitochondria or to the extracellular environment within living organisms, which are the two most important ROS sites in vivo. The targeting groups will be combined with a ROS sensitive moiety that reacts with a particular ROS to form a diagnostic product. Thus the extent of conversion of the starting probe to its diagnostic product will indicate the concentration of ROS in vivo in that compartment. All the probe molecules contain fixed charges, greatly enhancing their sensitivity of detection by mass spectrometry. Therefore the ratio or starting probe to diagnostic product will be assessed by extracting the probe and products from the animal and quantitating both by liquid chromatography tandem mass spectrometry (LC/MS/MS) in comparison to deuterated internal standards.

The average age of the UK population is rising and this presents major challenges to our society, its working practices and its health services. It is vital that we increase the proportion of people's lives when they are healthy and active (healthspan). As well as giving obvious benefit to individuals' health and happiness, this will also maximise the contribution of the most highly experienced people to society and the economy, while minimising their health costs. A better healthspan will also mean better availability of public services and lower taxation. The proposed research will provide the tools necessary to understand one of the main processes involved in ageing, i.e. oxidative stress. By understanding this key process, which appears to be involved in almost every age-related disease, we will be able to improve the UK healthspan through rationally designed interventions. The new tools to be developed are molecular probes that will be useful to scientists world-wide involved in the study of ageing, oxidative processes and diseases involving oxidative stress. Companies involved in supplying chemicals for biomedical research will benefit directly from selling these probes. The probes will include antioxidants targeted to extracellular regions and these may well have potential as cardiovascular drugs and for mitigation of the oxidative stress involved in autoimmune responses; diseases such as rheumatoid arthritis have immense personal and economic consequences to the UK. This will potentially benefit a UK SME involved in clinical-stage development of drug candidates and then a UK-based multinational pharmaceutical company. The income stream generated through IP and through supplying probes will also benefit the institutions involved in this research, which are major employers. The project will provide an excellent training for a PDRA in chemical biology, which is a cutting-edge area of science which needs to be developed in the UK to support biotechnology.