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

开发化学质谱探针来评估体内活性氧的产生

• 批准号: BB/I012923/1
• 负责人: Michael Murphy
• 金额: $ 3.9万
• 依托单位: MRC Centre Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI012923%2F1
• 关键词: Developing; chemical; mass; spectrometry; probes

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.

在我们的身体内，以及所有动物的身体内，我们呼吸的氧气会发生副反应，导致产生称为“活性氧”（ROS）的小分子。这些ROS是与衰老相关的氧化损伤的原因。事实上，有人认为，它们导致衰老过程本身和与之相关的条件，如神经退行性变。ROS也用于生物体内重要的信号通路。然而，虽然有很多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ROS的寿命很短，很难测量，所以虽然它们可以在试管中定量，但目前还不可能计算出生物体内存在多少ROS。由于这个困难，我们经常不确定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，使用分子探针，旨在进入线粒体，构成我们身体的细胞部分，被认为是ROS的主要来源。对线粒体的靶向依赖于简单的物理化学效应，由此能够穿过膜的亲脂性阳离子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。然后，起始分子与其产物的比率将给出分离的线粒体中以及全细胞和动物的线粒体中ROS的量的指示。类似的探针被设计为保留在细胞外，以便可以在细胞外环境中检测ROS，特别是在循环中。我们的方法必须非常敏感，以便评估生物体内发生的微小变化。为了促进这一点，探针的设计，使他们可以在复杂的生物混合物中检测选择性和灵敏度，即使在非常少量的存在，通过一种称为液相色谱-串联质谱技术。通过新探针结合靶向细胞或细胞外环境中的特定位置沿着其内置特征，允许通过质谱进行非常灵敏的检测，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，最重要的是对衰老过程本身的贡献。针对保持在细胞外的抗氧化剂也将被开发为一种干预措施，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。

项目成果

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

• DOI: 10.1038/nature13909
• 发表时间: 2014-11-20
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Chouchani, Edward T.;Pell, Victoria R.;Gaude, Edoardo;Aksentijevic, Dunja;Sundier, Stephanie Y.;Robb, Ellen L.;Logan, Angela;Nadtochiy, Sergiy M.;Ord, Emily N. J.;Smith, Anthony C.;Eyassu, Filmon;Shirley, Rachel;Hu, Chou-Hui;Dare, Anna J.;James, Andrew M.;Rogatti, Sebastian;Hartley, Richard C.;Eaton, Simon;Costa, Ana S. H.;Brookes, Paul S.;Davidson, Sean M.;Duchen, Michael R.;Saeb-Parsy, Kourosh;Shattock, Michael J.;Robinson, Alan J.;Work, Lorraine M.;Frezza, Christian;Krieg, Thomas;Murphy, Michael P.
• 通讯作者: Murphy, Michael P.

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes.

线粒体靶向质谱探针以检测乙二醇：对糖尿病的影响。

• DOI: 10.1016/j.freeradbiomed.2013.11.025
• 发表时间: 2014-02
• 期刊: FREE RADICAL BIOLOGY AND MEDICINE
• 影响因子: 7.4
• 作者: Pun, Pamela Boon Li;Logan, Angela;Darley-Usmar, Victor;Chacko, Balu;Johnson, Michelle S.;Huang, Guang W.;Rogatti, Sebastian;Prime, Tracy A.;Methner, Carmen;Krieg, Thomas;Fearnley, Ian M.;Larsen, Lesley;Larsen, David S.;Menger, Katja E.;Collins, Yvonne;James, Andrew M.;Kumar, G. D. Kishore;Hartley, Richard C.;Smith, Robin A. J.;Murphy, Michael P.
• 通讯作者: Murphy, Michael P.

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

• DOI: 10.1111/acel.12212
• 发表时间: 2014-08
• 期刊: Aging cell
• 影响因子: 7.8
• 作者: Logan A;Shabalina IG;Prime TA;Rogatti S;Kalinovich AV;Hartley RC;Budd RC;Cannon B;Murphy MP
• 通讯作者: Murphy MP

Expanding the palette of phenanthridinium cations.

• DOI: 10.1002/chem.201304241
• 发表时间: 2014-03-24
• 期刊: Chemistry (Weinheim an der Bergstrasse, Germany)
• 作者: Cairns AG;Senn HM;Murphy MP;Hartley RC
• 通讯作者: Hartley RC

Targeting mitochondria with small molecules: the preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide.

• DOI: 10.1007/978-1-4939-2288-8_3
• 发表时间: 2015-01-01
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Cairns, Andrew G;McQuaker, Stephen J;Hartley, Richard C
• 通讯作者: Hartley, Richard C