Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells

Abstract

Summary: The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry, suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified.For complete details on the use and execution of this protocol, please refer to Zellner et al. (2021).

Keywords

• Cell Biology
• Molecular Biology
• Protein Biochemistry
• Proteomics
• Mass Spectrometry