Mass spectrometry proteomics for studying mitostasis

Maintaining mitochondrial integrity and function is fundamental to cellular homeostasis. Cells rely on coordinated protein quality control (QC) systems-including intricate chaperone-protease networks, the ubiquitin-proteasome system, and cytosolic surveillance pathways-that together form a dynamic, cell-wide mitostasis network governing the import, folding, synthesis, and degradation of mitochondrial proteins. Disruption of mitochondrial homeostasis, for example, by impairing mitochondrial protein import, induces proteotoxic stress and contributes to human disease. Mass spectrometry (MS)-based proteomics has established itself as an indispensable method to dissect mitostasis at unprecedented depth by enabling systematic quantitative analysis of protein abundance, localization, interactions, stability, and dynamics. In this review, we highlight state-of-the-art MS technologies and multifaceted proteomics approaches used to study mitostasis on a proteome-wide level. These functional analysis approaches build on quantitative MS methods employing label-free, metabolic, and chemical labeling strategies, which allow precise tracking of proteome dynamics in response to different cellular conditions including stress. Spatial and interaction-based approaches, such as affinity purification-MS, proximity labeling, and complexome profiling, provide detailed insight into the organization and regulation of the complex mitochondrial organizing system, chaperone networks, and protein QC pathways. Furthermore, we discuss advanced methodologies such as nascent chain and dynamic proteomics strategies, which offer a proteome-wide comprehension of early stress responses and fast regulation. The skillful integration of temporal, spatial subcellular, interaction, nascent, and dynamic proteomics approaches now enables a systems-level assessment of mitostasis, paving the way for a holistic while nuanced understanding of this essential cellular process and the underlying molecular mechanisms.