Cairns Andrew G, McQuaker Stephen J, Murphy Michael P, Hartley Richard C
WestCHEM School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK.
Methods Mol Biol. 2015;1265:25-50. doi: 10.1007/978-1-4939-2288-8_3.
Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signalling and implicated in the oxidative damage associated with aging and a wide range of age-associated conditions including cardiovascular disease, neurodegeneration, and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15-MitoP and d15-MitoB as standard. Here we describe the synthesis of MitoB and MitoP and the deuterated standards necessary for this method of quantification.
小分子可以通过亲脂性的烷基三苯基鏻（TPP）基团在物理化学性质上靶向线粒体。一旦进入线粒体，TPP 共轭物可以直接检测或影响线粒体基质内的过程。或者，共轭物可以作为前药，通过内部或外部指令从 TPP 基团释放而被激活。可以设计出可用于任何细胞系、组织或整个生物体的小分子，实现时间控制，并以可逆的剂量依赖方式应用。一个例子是通过 MitoB 检测和定量整个活生物体线粒体中的过氧化氢。线粒体基质中产生的过氧化氢参与信号传导，并与衰老以及包括心血管疾病、神经退行性变和癌症在内的多种与年龄相关的病症所关联的氧化损伤有关。MitoB 在线粒体中积累，并被线粒体基质中的过氧化氢转化为外源性标志物 MitoP。经过一段时间的孵育后，根据 MitoP 与 MitoB 的比例确定过氧化氢浓度，该比例通过使用 d15-MitoP 和 d15-MitoB 作为标准品的质谱法测定。在这里，我们描述了 MitoB 和 MitoP 的合成以及这种定量方法所需的氘代标准品。
