Optimising complexome profiling for systematic exploration of DNA-/RNA-protein complexes in human mitochondria

Here we aimed to improve native gel electrophoresis-based complexome profiling (CP) method for examination of mitochondrial (mt)DNA-/RNA-protein complexes. We compared the performance of Blue Native PAGE (BNE) and high resolution Clear Native PAGE (hrCNE). We found that hrCNE much better preserves the integrity of mtDNA-/RNA-protein complexes.To further improve the detection of mtDNA-binding proteins, we treated solubilized mitochondrial samples with DNase I prior to separation on hrCNE or BNE. To characterize the detected by hrCNE mtRNA-protein complexes, we performed CP with samples treated with RNase A. To validate the utility of the method, we performed CP using mitochondria isolated from cells treated with ethidium bromide as well as from cells recovering after this treatment. Our study not only helps to validate and unveil proteins involved in mitochondrial DNA- and RNA-related processes, but also offers a convenient and systematic way for analysing these interactions that can be used equally well for the investigation of the nucleus and other DNA-/RNA-containing cell compartments.

本研究旨在优化基于天然凝胶电泳的复合物谱分析（complexome profiling, CP）方法，以用于检测线粒体DNA/RNA蛋白复合物。我们比较了蓝色天然聚丙烯酰胺凝胶电泳（Blue Native PAGE, BNE）与高分辨率透明天然聚丙烯酰胺凝胶电泳（high resolution Clear Native PAGE, hrCNE）的性能，结果发现hrCNE能够更好地维持线粒体DNA/RNA蛋白复合物的完整性。为进一步提升线粒体DNA结合蛋白的检测效果，我们在通过hrCNE或BNE进行分离前，先用脱氧核糖核酸酶I（DNase I）处理可溶性线粒体样品。为表征通过hrCNE检测到的线粒体RNA蛋白复合物，我们对经核糖核酸酶A（RNase A）处理的样品开展了复合物谱分析。为验证该方法的实用性，我们分别使用经溴化乙锭（ethidium bromide）处理的细胞以及经该处理后恢复的细胞所分离的线粒体开展了复合物谱分析。本研究不仅有助于验证并揭示参与线粒体DNA及RNA相关过程的蛋白质，同时还提供了一种便捷且系统的分析此类相互作用的方法，该方法同样可用于研究细胞核及其他含有DNA/RNA的细胞区室。