Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites.
Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites.
复制标题
DOI：
10.1038/nprot.2017.104
复制
发表时间：
2017-10
期刊：
影响因子：
14.8
通讯作者：
Sabatini DM
中科院分区：
文献类型：
--
作者：
Chen WW;Freinkman E;Sabatini DM
线粒体进行许多对细胞内稳态至关重要的代谢反应。在这里，我们提出了一个方案，询问线粒体代谢产物和测量其基质浓度。我们的工作流程利用高亲和力磁性免疫捕获，在约12分钟内从均质化的哺乳动物细胞中快速纯化HA标记的线粒体。这些线粒体用甲醇和水提取。液相色谱和质谱法（LC/MS）用于使用真实的代谢物标准品和同位素标记的内标物确定线粒体代谢物的身份和摩尔量，同时通过免疫印迹、完整细胞的共聚焦显微镜和体积分析确定相应的线粒体基质体积。一旦获得所有值，将基质体积与上述摩尔量结合，以计算线粒体代谢物的基质浓度。与替代方法相比，缩短了分离时间并提高了分离线粒体的纯度，这种LC/MS兼容的工作流程允许对线粒体代谢物进行稳健的分析，并作为可推广到其他哺乳动物细胞器研究的策略。一旦所有必要的试剂都已准备好，就可以在一周内完成线粒体代谢物基质浓度的定量。
Mitochondria carry out numerous metabolic reactions that are critical for cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow utilizes high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in ~12 minutes. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically-labeled internal standards, while the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved purity of isolated mitochondria compared to alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all of the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.
登录
查看更多内容
影响因子：
2.5
作者：
Schindelin J;Rueden CT;Hiner MC;Eliceiri KW
通讯作者：
Eliceiri KW
影响因子：
5.2
作者：
Cacciatore S;Loda M
通讯作者：
Loda M
游离氨基酸的线粒体池反映线粒体DNA编码蛋白的组成：指示蛋白质合成后翻译后质量控制。
影响因子：
4
作者：
Ross-Inta C;Tsai CY;Giulivi C
通讯作者：
Giulivi C
影响因子：
3.7
作者：
Roede JR;Park Y;Li S;Strobel FH;Jones DP
通讯作者：
Jones DP
影响因子：
14.8
作者：
Frezza, Christian;Cipolat, Sara;Scorrano, Luca
通讯作者：
Scorrano, Luca