Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge. Herein, we present a comprehensive subcellular fractionation, characterization, and metabolome analysis strategy. First, six subcellular fractions including nuclei, mitochondria, lysosomes, peroxisomes, microsomes, and cytoplasm were generated from a single aliquot of liver homogenate. Then, a dansyl-labeling-assisted liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for measuring 151 amino/phenol- or carboxyl-containing metabolites in the subcellular fractions was established and validated. Last, the strategy was applied to a rat model of carbon tetrachloride (CCl₄)-induced acute liver injury (ALI). The metabolic profile of individual organelles was compared with that of the liver. Interestingly, many unique changes were observed specifically in organelles, while the liver failed to capture these changes. This result indicates that metabolic investigation at the tissue level might lead to erroneous results due to the leveling effect. Our study demonstrates a feasible approach for the broad-spectrum-targeted metabolic profiling of multi-subcellular fractions, which can be of great use in driving our further understanding of intracellular metabolic activities in various physical and pathological conditions.

中文翻译：

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使用基于丹磺酰标记的 LC-MS/MS 进行多亚细胞代谢组分析的高覆盖率策略

亚细胞区室化确保真核生命中有序、高效的细胞内代谢活动。亚细胞代谢组的研究可以提供对细胞生物学活动的深入了解。然而，多亚细胞代谢谱的灵敏测量仍然是一个重大挑战。在此，我们提出了全面的亚细胞分离、表征和代谢组分析策略。首先，从一份肝脏匀浆中产生六种亚细胞组分，包括细胞核、线粒体、溶酶体、过氧化物酶体、微粒体和细胞质。然后，建立并验证了丹磺酰标记辅助液相色谱-串联质谱 (LC-MS/MS) 方法，用于测量亚细胞组分中 151 种含氨基/酚或羧基的代谢物。最后的，₄ )引起的急性肝损伤(ALI)。将单个细胞器的代谢特征与肝脏的代谢特征进行比较。有趣的是，在细胞器中观察到了许多独特的变化，而肝脏未能捕获这些变化。该结果表明，由于均化效应，组织水平的代谢研究可能会导致错误的结果。我们的研究展示了一种对多亚细胞组分进行广谱靶向代谢分析的可行方法，这对于推动我们进一步了解各种物理和病理条件下的细胞内代谢活动非常有用。