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

在我们和所有动物体内，我们呼吸的氧气会发生副反应，产生一种叫做活性氧（ROS）的小分子。这些活性氧负责与衰老相关的氧化损伤。事实上，有人认为它们会导致衰老过程本身以及与之相关的神经变性等病症。活性氧也用于生物体内重要的信号通路。然而，虽然有大量的间接证据表明活性氧在生物体内的重要性，但它们的作用程度也存在相当大的不确定性。这是因为活性氧的寿命很短，很难测量，所以虽然它们可以在试管中量化，但目前还不可能计算出生物体中存在多少活性氧。由于这一困难，我们常常不确定ROS在一系列重要的生物过程中有多重要，如衰老、疾病、生长和癌症。为了克服这一困难，我们将开发一种新的方法，利用分子探针进入线粒体，线粒体是构成我们身体的细胞的一部分，被认为是ROS的主要来源。靶向线粒体依赖于一种简单的物理化学效应，即亲脂性阳离子能够穿过膜在线粒体基质中积累。一旦到达那里，探针分子将与ROS反应形成独特的产物。起始分子与其产物的比例将指示分离线粒体和整个细胞和动物线粒体中ROS的数量。类似的探针被设计留在细胞外，这样就可以在细胞外环境中检测到ROS，特别是在循环中。我们的方法必须非常敏感，以便评估生物体内部发生的微小变化。为了促进这一点，这些探针被设计成可以通过一种称为液相色谱-串联质谱法的技术，在复杂的生物混合物中选择性地、灵敏地检测，即使是在非常少量的情况下。通过新探针的组合靶向细胞或细胞外环境中的特定位置，以及它们内置的特性，允许通过质谱进行非常敏感的检测，我们将能够测量实验动物中的ROS，从而首次计算出ROS对许多重要生物过程的详细贡献，最重要的是衰老过程本身。针对留在细胞外的抗氧化剂也将被开发，作为一种干预手段，使我们能够评估循环和细胞外基质中氧化应激的具体贡献如何影响整个衰老过程。

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.