CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2021 Aug 17;93(32):11099-11107. doi: 10.1021/acs.analchem.1c01101. Epub 2021 Aug 4.
As a vital hub, a mitochondrion houses metabolic pathways that play important roles in cellular physiology. Aberrant metabolites occurring in mitochondria are closely associated with the emergence and progression of various mitochondria-related diseases. Therefore, a simple and versatile approach to efficiently purify intact mitochondria is urgently needed to precisely and comprehensively characterize the composition and abundance of the mitochondrial metabolome in different physiological and pathological states. In this work, novel immunoaffinitive magnetic composites MagG@PD@Avidin@TOM20 were prepared to achieve highly selective isolation of intact mitochondria from three different hepatocytes (LO2, HepG2, and Huh7). The prepared composites inherit combined merits, including strong magnetic responsiveness, excellent stability, and specific and high affinity between antibody TOM20 and mitochondrial outer membrane protein. These mitochondria attached on MagG@PD@Avidin@TOM20 were characterized by the western blot and fluorescence microscopy to confirm their purity and integrity, which are vital for reliable mitochondrial metabolic analysis. Subsequently, ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based untargeted metabolomics analysis was conducted to characterize the metabolomes in the immunopurified mitochondria and whole cells. Notably, the metabolite profiles of whole cells and mitochondria including itaconic acid, acetylcarnitine, malic acid, etc., were significantly different. These data underscore the importance of determining metabolites at the mitochondrial level, which would supplement us new knowledge at the subcellular level.
作为一个重要的枢纽，线粒体内部的代谢途径在细胞生理学中起着重要作用。线粒体中异常的代谢物与各种与线粒体相关疾病的发生和发展密切相关。因此，迫切需要一种简单而通用的方法来有效地纯化完整的线粒体，以便在不同的生理和病理状态下精确和全面地表征线粒体代谢组的组成和丰度。在这项工作中，我们制备了新型免疫亲和磁性复合材料 MagG@PD@Avidin@TOM20，用于从三种不同的肝细胞（LO2、HepG2 和 Huh7）中高度选择性地分离完整的线粒体。所制备的复合材料继承了联合优点，包括强磁响应性、优异的稳定性以及抗体 TOM20 和线粒体外膜蛋白之间的特异性和高亲和力。通过 Western blot 和荧光显微镜对附着在 MagG@PD@Avidin@TOM20 上的线粒体进行了表征，以确认其纯度和完整性，这对于可靠的线粒体代谢分析至关重要。随后，我们进行了基于超高效液相色谱-高分辨率质谱的非靶向代谢组学分析，以表征免疫纯化的线粒体和整个细胞中的代谢组。值得注意的是，整个细胞和线粒体的代谢物图谱，包括衣康酸、乙酰肉碱、苹果酸等，有明显的差异。这些数据强调了在亚细胞水平上确定代谢物的重要性，这将为我们提供新的亚细胞水平的知识。
