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

Autor: Susanne Zellner, Karsten Nalbach, Christian Behrends
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: STAR Protocols, Vol 2, Iss 2, Pp 100506- (2021)
Druh dokumentu: article
ISSN: 2666-1667
DOI: 10.1016/j.xpro.2021.100506
Popis: 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).
Databáze: Directory of Open Access Journals