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Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Significance We use a protein labeling technique based on an engineered ascorbate peroxidase (APEX) to map the proteome of the mitochondrial matrix in live tissues. The approach allows us to establish MitoMax, a comprehensive database providing a high-quality inventory of Drosophila mitochondrial proteins with subcompartmental annotation. We demonstrate that APEX labeling is effective in vivo and provides an opportunity to characterize subcellular proteomes in specific cell types and in different physiological conditions. Given the interest in defining the mitochondrial proteome in different physiological conditions and tissues, our analysis provides a resource for systematic functional analyses of mitochondria that will in particular facilitate investigation of mitochondrial diseases. Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.