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

Authors
  • Zellner, Susanne1
  • Nalbach, Karsten1
  • Behrends, Christian1
  • 1 Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, Feodor-Lynen Strasse 17, 81377 Munich, Germany. , (Germany)
Type
Published Article
Journal
STAR protocols
Publication Date
Jun 18, 2021
Volume
2
Issue
2
Pages
100506–100506
Identifiers
DOI: 10.1016/j.xpro.2021.100506
PMID: 33997820
Source
Medline
Keywords
Language
English
License
Unknown

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). © 2021 The Author(s).

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