The study by Sickmann et al. (2003) provides a comprehensive analysis of the proteome of Saccharomyces cerevisiae mitochondria. The researchers isolated highly pure yeast mitochondria and used multiple separation methods, including 2D PAGE, multidimensional liquid chromatography, and 1D PAGE, followed by tandem MS analysis. From over 20 million MS spectra, 750 different proteins were identified, representing approximately 90% of all known mitochondrial proteins. The identified proteins include components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and stable mitochondria-encoded proteins. The study also highlights the function of a quarter of the identified proteins, which remains unknown. The mitochondrial proteome will serve as a valuable resource for understanding new mitochondrial functions and characterizing mitochondrial diseases. The authors emphasize the importance of using authentic, nontagged, and nonoverexpressed proteins for reliable localization studies, as tagging approaches can lead to mislocalization. The study's high sensitivity and comprehensive coverage of the mitochondrial proteome are highlighted, with the identification of presequences on the protein level being a notable finding. The functional classification of the identified proteins reveals diverse roles in energy metabolism, genome maintenance, metabolism, protein translocation, and signaling.The study by Sickmann et al. (2003) provides a comprehensive analysis of the proteome of Saccharomyces cerevisiae mitochondria. The researchers isolated highly pure yeast mitochondria and used multiple separation methods, including 2D PAGE, multidimensional liquid chromatography, and 1D PAGE, followed by tandem MS analysis. From over 20 million MS spectra, 750 different proteins were identified, representing approximately 90% of all known mitochondrial proteins. The identified proteins include components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and stable mitochondria-encoded proteins. The study also highlights the function of a quarter of the identified proteins, which remains unknown. The mitochondrial proteome will serve as a valuable resource for understanding new mitochondrial functions and characterizing mitochondrial diseases. The authors emphasize the importance of using authentic, nontagged, and nonoverexpressed proteins for reliable localization studies, as tagging approaches can lead to mislocalization. The study's high sensitivity and comprehensive coverage of the mitochondrial proteome are highlighted, with the identification of presequences on the protein level being a notable finding. The functional classification of the identified proteins reveals diverse roles in energy metabolism, genome maintenance, metabolism, protein translocation, and signaling.