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

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

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 autop...

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Bibliographic Details
Main Authors: Susanne Zellner, Karsten Nalbach, Christian Behrends
Format: Article
Language:English
Published: Elsevier 2021-06-01
Series:STAR Protocols
Subjects:
Cell Biology
Molecular Biology
Protein Biochemistry
Proteomics
Mass Spectrometry
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166721002136
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spelling doaj-046bb2d1a8a54597a74d18c39dfb15a12021-06-21T04:25:27ZengElsevierSTAR Protocols2666-16672021-06-0122100506Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cellsSusanne Zellner0Karsten Nalbach1Christian Behrends2Munich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, Feodor-Lynen Strasse 17, 81377 Munich, GermanyMunich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, Feodor-Lynen Strasse 17, 81377 Munich, GermanyMunich Cluster for Systems Neurology (SyNergy), Medical Faculty, Ludwig-Maximilians-University München, Feodor-Lynen Strasse 17, 81377 Munich, Germany; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S2666166721002136Cell BiologyMolecular BiologyProtein BiochemistryProteomicsMass Spectrometry
collection DOAJ
language English
format Article
sources DOAJ
author Susanne Zellner
Karsten Nalbach
Christian Behrends
spellingShingle Susanne Zellner
Karsten Nalbach
Christian Behrends
Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
STAR Protocols
Cell Biology
Molecular Biology
Protein Biochemistry
Proteomics
Mass Spectrometry
author_facet Susanne Zellner
Karsten Nalbach
Christian Behrends
author_sort Susanne Zellner
title Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
title_short Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
title_full Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
title_fullStr Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
title_full_unstemmed Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
title_sort autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells
publisher Elsevier
series STAR Protocols
issn 2666-1667
publishDate 2021-06-01
description 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).
topic Cell Biology
Molecular Biology
Protein Biochemistry
Proteomics
Mass Spectrometry
url http://www.sciencedirect.com/science/article/pii/S2666166721002136
work_keys_str_mv AT susannezellner autophagosomecontentprofilingusingproximitybiotinylationproteomicscoupledtoproteasedigestioninmammaliancells
AT karstennalbach autophagosomecontentprofilingusingproximitybiotinylationproteomicscoupledtoproteasedigestioninmammaliancells
AT christianbehrends autophagosomecontentprofilingusingproximitybiotinylationproteomicscoupledtoproteasedigestioninmammaliancells
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