Le Xuyen, Millar A Harvey, Lee Chun Pong
ARC Centre of Excellence in Plant Energy Biology, Bayliss Building M316, University of Western Australia, Crawley, WA, Australia.
Methods Mol Biol. 2022;2363:85-100. doi: 10.1007/978-1-0716-1653-6_8.
Transport of tricarboxylic acid (TCA) cycle substrates across mitochondrial membranes and their subsequent oxidative decarboxylation in the matrix provide reductants for respiration-coupled ATP synthesis. These processes are typically assessed together through the ability of mitochondria to consume oxygen or release carbon dioxide, however, this approach fails to assess or separate the complexity of transport and the subsequent metabolism of substrates and products. In this chapter, we provide a strategy for simultaneously measuring substrate transport and utilization by isolated mitochondria using a mass spectrometry-based technique. The results of cofeeding of isolated mitochondria with unlabeled malate and uniformly labeled pyruvate is used as an example. Mitochondria fed with substrates are separated from the extramitochondrial space by centrifugation through a single layer of silicone oil. Analysis of mitochondrial pellet and reaction supernatant enable quantitation of substrate import and product export. This method also allows an estimation of the contribution of different enzymatic pathways to the formation of a specific product. This assay opens opportunities to verify carrier functions in organello and to identify the substrate preferences of mitochondrial transporters of unknown function using targeted and/or untargeted metabolomics approaches.
三羧酸（TCA）循环底物穿过线粒体膜的转运及其随后在基质中的氧化脱羧作用为呼吸偶联的ATP合成提供还原剂。这些过程通常通过线粒体消耗氧气或释放二氧化碳的能力来共同评估，然而，这种方法无法评估或区分转运的复杂性以及底物和产物随后的代谢情况。在本章中，我们提供了一种使用基于质谱的技术同时测量分离的线粒体底物转运和利用情况的策略。以未标记的苹果酸和均匀标记的丙酮酸共同喂养分离的线粒体的结果为例。通过单层硅油离心将摄取底物的线粒体与线粒体外空间分离。对线粒体沉淀和反应上清液的分析能够定量底物的摄取和产物的输出。该方法还可以估计不同酶促途径对特定产物形成的贡献。这种测定方法为在细胞器中验证载体功能以及使用靶向和/或非靶向代谢组学方法鉴定未知功能的线粒体转运体的底物偏好提供了机会。
