Mitochondria are multifaceted organelles with key roles in anabolic and catabolic metabolism, bioenergetics, cellular signalling and nutrient sensing, and programmed cell death processes. Their diverse functions are enabled by a sophisticated set of protein components encoded by the nuclear and mitochondrial genomes. The extent and complexity of the mitochondrial proteome remained unclear for decades. This began to change 20 years ago when, driven by the emergence of mass spectrometry-based proteomics, the first draft mitochondrial proteomes were established. In the ensuing decades, further technological and computational advances helped to refine these ‘maps’, with current estimates of the core mammalian mitochondrial proteome ranging from 1,000 to 1,500 proteins. The creation of these compendia provided a systemic view of an organelle previously studied primarily in a reductionist fashion and has accelerated both basic scientific discovery and the diagnosis and treatment of human disease. Yet numerous challenges remain in understanding mitochondrial biology and translating this knowledge into the medical context. In this Roadmap, we propose a path forward for refining the mitochondrial protein map to enhance its discovery and therapeutic potential. We discuss how emerging technologies can assist the detection of new mitochondrial proteins, reveal their patterns of expression across diverse tissues and cell types, and provide key information on proteoforms. We highlight the power of an enhanced map for systematically defining the functions of its members. Finally, we examine the utility of an expanded, functionally annotated mitochondrial proteome in a translational setting for aiding both diagnosis of mitochondrial disease and targeting of mitochondria for treatment.

线粒体是多方面的细胞器，在合成代谢和分解代谢、生物能学、细胞信号传导和营养传感以及程序性细胞死亡过程中发挥着关键作用。它们的多种功能是由核和线粒体基因组编码的一组复杂的蛋白质成分实现的。几十年来，线粒体蛋白质组的范围和复杂性仍不清楚。20 年前，这种情况开始发生变化，当时，在基于质谱的蛋白质组学出现的推动下，建立了第一份线粒体蛋白质组草稿。在接下来的几十年里，进一步的技术和计算进步帮助完善了这些“图谱”，目前对核心哺乳动物线粒体蛋白质组的估计范围为 1,000 至 1,500 个蛋白质。这些概要的创建提供了以前主要以还原论方式研究的细胞器的系统视图，并加速了基础科学发现以及人类疾病的诊断和治疗。然而，在理解线粒体生物学并将这些知识转化为医学背景方面仍然存在许多挑战。在本路线图中，我们提出了一条完善线粒体蛋白质图谱以增强其发现和治疗潜力的前进道路。我们讨论新兴技术如何帮助检测新的线粒体蛋白，揭示它们在不同组织和细胞类型中的表达模式，并提供有关蛋白质形式的关键信息。我们强调增强地图在系统地定义其成员职能方面的力量。最后，我们研究了扩展的、功能注释的线粒体蛋白质组在翻译环境中的效用，以帮助诊断线粒体疾病和靶向线粒体进行治疗。