Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs.
Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs.
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DOI：
10.1038/s41598-020-74718-1
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发表时间：
2020-10-19
影响因子：
4.6
通讯作者：
Fisher-Wellman KH
中科院分区：
文献类型：
--
作者：
McLaughlin KL;Hagen JT;Coalson HS;Nelson MAM;Kew KA;Wooten AR;Fisher-Wellman KH
人类疾病病理生理学通常涉及细胞和亚细胞水平的代谢破坏。分离的线粒体是一个强大的模型，用于分离整体细胞的变化从内在的线粒体改变。然而，用于分离线粒体的常见实验室实践（例如，差速离心）通常导致具有可变线粒体纯度的细胞器制备物。为了克服这个问题，我们开发了一种基于质谱的方法，定量评估样品特异性线粒体富集百分比。然后使用样品特异性线粒体富集将线粒体功能的各种生化读数校正为“固定”量的线粒体蛋白，从而允许在多个小鼠组织（例如，心脏、棕色脂肪、肾脏、肝脏）。我们的研究结果支持使用靶向的nLC-MS/MS作为一种方法，以定量线粒体富集在每个样本的基础上，允许无偏比较的功能参数群体之间的线粒体分离的代谢不同的组织。这种方法可以很容易地应用于多个实验环境中，其中线粒体网络中的内在变化被怀疑驱动给定的生理或病理生理结果。
Human disease pathophysiology commonly involves metabolic disruption at both the cellular and subcellular levels. Isolated mitochondria are a powerful model for separating global cellular changes from intrinsic mitochondrial alterations. However, common laboratory practices for isolating mitochondria (e.g., differential centrifugation) routinely results in organelle preparations with variable mitochondrial purity. To overcome this issue, we developed a mass spectrometry-based method that quantitatively evaluates sample-specific percent mitochondrial enrichment. Sample-specific mitochondrial enrichment was then used to correct various biochemical readouts of mitochondrial function to a ‘fixed’ amount of mitochondrial protein, thus allowing for intrinsic mitochondrial bioenergetics, relative to the underlying proteome, to be assessed across multiple mouse tissues (e.g., heart, brown adipose, kidney, liver). Our results support the use of mitochondrial-targeted nLC-MS/MS as a method to quantitate mitochondrial enrichment on a per-sample basis, allowing for unbiased comparison of functional parameters between populations of mitochondria isolated from metabolically distinct tissues. This method can easily be applied across multiple experimental settings in which intrinsic shifts in the mitochondrial network are suspected of driving a given physiological or pathophysiological outcome.