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

STAR Protoc. 2021 Apr 27;2(2):100506. doi: 10.1016/j.xpro.2021.100506. PMID: 33997820; PMCID: PMC8102175.

Authors/Editors: Zellner S, Nalbach K, Behrends C.
Publication Date: 2021

Abstract

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).

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