The proteome of Saccharomyces cerevisiae mitochondria was comprehensively analyzed, identifying 750 different proteins, representing approximately 90% of all mitochondrial proteins. This study used multiple methods, including 2D PAGE, tandem MS, and multidimensional peptide separation, to isolate and analyze highly pure yeast mitochondria. The identified proteins included all known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and stable mitochondrial-encoded proteins. The results indicate that the mitochondrial proteome provides a valuable database for understanding mitochondrial and mitochondria-associated functions and diseases. Mitochondria are central to many cellular processes, including bioenergetics, apoptosis, and metabolism. However, only about 50-60% of mitochondrial proteins have been identified so far, limiting our understanding of their functions. The yeast genome was the first fully sequenced eukaryotic genome, and it serves as a model organism for studying protein functions and cellular pathways. The study identified 750 proteins, with 436 being known mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The high purity of the mitochondria used in the study suggests that many proteins previously found in other compartments are actually mitochondrial or mitochondria-associated proteins. The study also revealed that some proteins have dual localization in mitochondria and other cellular compartments. The yeast mitochondrial proteome covers 92% of the known mitochondrial proteins listed in the MITOP yeast database. The study also identified 65 subunits of the yeast mitochondrial ribosome, covering 89% of the known subunits. The findings highlight the importance of the yeast mitochondrial proteome in understanding mitochondrial functions and diseases. The study used various methods to ensure the accuracy of the results, including the use of multiple separation techniques and database searches. The results indicate that the mitochondrial proteome is a comprehensive resource for further research into mitochondrial biology.The proteome of Saccharomyces cerevisiae mitochondria was comprehensively analyzed, identifying 750 different proteins, representing approximately 90% of all mitochondrial proteins. This study used multiple methods, including 2D PAGE, tandem MS, and multidimensional peptide separation, to isolate and analyze highly pure yeast mitochondria. The identified proteins included all known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and stable mitochondrial-encoded proteins. The results indicate that the mitochondrial proteome provides a valuable database for understanding mitochondrial and mitochondria-associated functions and diseases. Mitochondria are central to many cellular processes, including bioenergetics, apoptosis, and metabolism. However, only about 50-60% of mitochondrial proteins have been identified so far, limiting our understanding of their functions. The yeast genome was the first fully sequenced eukaryotic genome, and it serves as a model organism for studying protein functions and cellular pathways. The study identified 750 proteins, with 436 being known mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The high purity of the mitochondria used in the study suggests that many proteins previously found in other compartments are actually mitochondrial or mitochondria-associated proteins. The study also revealed that some proteins have dual localization in mitochondria and other cellular compartments. The yeast mitochondrial proteome covers 92% of the known mitochondrial proteins listed in the MITOP yeast database. The study also identified 65 subunits of the yeast mitochondrial ribosome, covering 89% of the known subunits. The findings highlight the importance of the yeast mitochondrial proteome in understanding mitochondrial functions and diseases. The study used various methods to ensure the accuracy of the results, including the use of multiple separation techniques and database searches. The results indicate that the mitochondrial proteome is a comprehensive resource for further research into mitochondrial biology.