Bio Protoc. 2026 Feb 20. 16(4):
e5605
The cellular compartments of eukaryotic cells are defined by their specific protein compositions. Different strategies are used for the identification of the subcellular proteomes, such as fractionation by differential centrifugation of cellular extracts. The localization of mitochondrial proteins is particularly challenging, as mitochondria consist of two membranes of different protein composition and two aqueous subcompartments, the intermembrane space (IMS) and the matrix. Previous studies identified subcompartment-specific proteomes by using combinations of hypotonic swelling and protease digestion followed by mass spectrometry. Here, we present an alternative, more unbiased method to identify the proteomes of mitochondrial subcompartments by use of an improved ascorbate peroxidase (APEX2) that is targeted to the IMS and the matrix. This method allows the subcompartment-specific labeling of proteins in mitochondria isolated from cells of the baker's yeast Saccharomyces cerevisiae, followed by their purification on streptavidin beads. With this method, the proteins located in the different mitochondrial subcompartments of yeast cells can be efficiently and comprehensively identified. Key features • Coverage of ~75% of previous combined annotated mitochondrial proteome studies with high confidence in sub-localization probabilities. • Provides detailed steps from starting culture to MS sample preparation, including the isolation of mitochondria. • Allows for easy adaptations to compare different conditions and treatments. • The whole experiment requires at least five days to complete.
Keywords: APEX2; Mitochondria isolation; Mitochondrial proteome; Proximity labeling; S. cerevisiae; Sub-localization