Introduction

Mitochondria represent an important milieu for studying the pathogenesis of several major diseases. The need for organelle-level metabolic resolution exists, as mitochondrial/cytosolic metabolites are often diluted beyond detection limits in complex samples. Compartment-specific studies are still hindered by the lack of efficient, cost-effective fractioning methods—applicable to laboratories of all financial/analytical standing.

Objectives

We established a novel mitochondrial/cytosolic purification pipeline for complimentary GC-TOF–MS and ¹H-NMR metabolomics using robust, commercially available fractionation strategies.

Methods

Magnetic based mitochondria isolation kits (MACS) were adapted for this purpose, accompanied by cytosolic filtering. Yield was assessed through the percentage recovery of citrate synthase (CS; a mitochondrial marker), purity by immunoblotting against compartment-specific proteins and integrity interrogated through the respiratory coupling ratio (RCR). The effects of the kit-based buffers on MS/NMR analyses of pure metabolite standards were evaluated. Finally, biological applicability to mammalian disease models was shown using Ndufs4 mouse brain tissue.

Results

With minor modifications, MACS produced around 60% more mitochondria compared to a differential centrifugation method. Less than 15% of lysosomal LAMP-2 protein was found in the MACS isolates, confirming relative purity—while RCR’s above 6 indicate sufficient mitochondrial integrity. The filtering approach effectively depleted mitochondria from the cytosolic fraction, as indicated by negligible Hsp60 and CS levels. Our GC–MS pilot yielded 60–70 features per fraction, while NMR analyses could quantify 6–10 of the most abundant compounds in each fraction.

Conclusion

This study provides a simple and flexible solution for mitochondrial and cytosolic metabolomics in animal model tissues, towards large-scale application of such methodologies in disease research.

中文翻译：

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新型线粒体和胞质纯化管线，用于哺乳动物疾病模型组织中的区室特异性代谢组学。

介绍

线粒体代表了研究几种主要疾病发病机理的重要环境。由于复杂样品中的线粒体/胞质代谢物通常被稀释到检测限以外，因此需要细胞器水平的代谢分解。由于缺乏适用于所有财务/分析领域的实验室的高效，经济高效的分馏方法，仍然阻碍了针对室的研究。

目标

我们建立了一种新颖的线粒体/胞质纯化流水线，以使用强大的市售分离策略，用于互补的GC-TOF-MS和¹ H-NMR代谢组学。

方法

基于磁性的线粒体分离试剂盒（MACS）为此目的进行了改进，并进行了胞质过滤。通过柠檬酸合酶（CS；线粒体标记）的回收率，通过针对特定区室蛋白的免疫印迹和通过呼吸耦合比（RCR）询问完整性的纯度来评估产量。评估了基于试剂盒的缓冲液对纯代谢产物标准品的MS / NMR分析的影响。最后，使用Ndufs4小鼠脑组织显示了对哺乳动物疾病模型的生物学适用性。

结果

稍加修改，与差分离心法相比，MACS产生的线粒体增加了约60％。在MACS分离物中发现不到15％的溶酶体LAMP-2蛋白，证实了其相对纯度-而RCR高于6则表明线粒体具有足够的完整性。Hsp60和CS水平可忽略不计，该过滤方法有效地从线粒体部分清除了线粒体。我们的GC-MS试点产品每个馏分可产生60-70个特征，而NMR分析可量化每个馏分中6-10个最丰富的化合物。

结论

这项研究为动物模型组织中的线粒体和胞质代谢组学提供了一种简单而灵活的解决方案，以在疾病研究中大规模应用这种方法。