A high-confidence plant mitochondrial proteome via protein correlation profiling.

Plant mitochondria share many features with animal mitochondria but also possess unique traits, including presence of both circular and linear mitochondrial DNA, RNA editing, a branched electron transport chain, and metabolic pathways supporting photosynthesis. An accurate mitochondrial proteome is crucial for studying these conserved and lineage-specific functions. Here, we introduce a novel approach to defining plant mitoproteomes that combines serial mitochondrial enrichment, mass spectrometry, and protein correlation profiling. This approach allows us to experimentally define a mitochondrial protein inventory of 1,462 proteins with high sensitivity and specificity. Integration of our experimental inventory with literature and microscopy validation yields the Arabidopsisthaliana MitoCarta (AtMitoCarta) atlas of 1,609 mitochondrial proteins, including 236 newly defined proteins and 147 proteins of unknown functions. A comparative analysis across eukaryotic organisms reveals that 15% of mitochondrial protein families are plant specific, including expanded pentatricopeptide repeat proteins. The AtMitoCarta inventory and the experimental workflow applicable to wild-type plants will enable comparative studies of plant mitochondria.