Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

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.Specialized biological processes are carried out in specific organelles and subcellular compartments. For example, mitochondria are the site of oxidative respiration, neurons pass electrical or chemical signals to others through synapses, and apical and basolateral domains of epithelial cells are critical for their polarized functions. Understanding how these structures underlie specialized functions requires the comprehensive identification of proteins within spatially defined cellular domains.A common strategy to study the localization of a particular protein is to generate green fluorescent protein (GFP) fusion proteins. However, it is time-consuming and labor-intensive to investigate protein localization at a large scale using GFP tagging, especially in vivo. Therefore, highly sensitive mass spectrometry (MS) approaches have been developed to systematically characterize the proteome of subcellular compartments. However, using MS approaches to characterize the proteome of subcellular domains has been limited by purification methods and is commonly associated with numerous false positives and false negatives due to contamination and loss of components during purification, respectively. For example, mitochondria are composed of an outer membrane and an inner membrane, generating two subcompartmental regions: the intermembrane space and the matrix located within the inner membrane. Because the ultrastructure of mitochondria is often disrupted during isolation processes, the isolation of specific subcompartmental regions of mitochondria is prone to contamination.Recently, a method based on an engineered ascorbate peroxidase (APEX) has been developed and shown to function in cultured mammalian cells for proteomic mapping (1). Upon activation, the APEX enzyme turns a biotin-phenol substrate into a highly reactive radical that covalently tags neighboring proteins on electron-rich amino acids such as tyrosine. Biotinylated endogenous proteins can then be isolated and identified by MS. Thus, APEX labeling can be applied to bypass organelle purification steps, offering an alternative approach for systematic proteomic characterization in live cells. Here we report that the approach can be applied to characterize the subcellular proteome in live tissues and map the mitochondrial matrix proteome of Drosophila muscle. In addition to characterizing a number of uncharacterized putative mitochondrial proteins, we establish MitoMax, a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation.