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

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).

SUBMITTER: Zellner S

PROVIDER: S-EPMC8102175 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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

Zellner Susanne S Nalbach Karsten K Behrends Christian C

STAR protocols 20210427 2

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, ...[more]

PMID: 33997820

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