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

Zellner, Susanne; Nalbach, Karsten und Behrends, Christian (2021): Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells. In: Star Protocols, Bd. 2, Nr. 2, 100506

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 pro-teinase K digestion of cell homogenates, proteins enriched in membrane -pro-tected 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).

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin > Munich Cluster for Systems Neurology (SyNergy)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 390857198