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Off-the-shelf proximity biotinylation for interaction proteomics
Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translat...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373943/ https://www.ncbi.nlm.nih.gov/pubmed/34408139 http://dx.doi.org/10.1038/s41467-021-25338-4 |
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author | Santos-Barriopedro, Irene van Mierlo, Guido Vermeulen, Michiel |
author_facet | Santos-Barriopedro, Irene van Mierlo, Guido Vermeulen, Michiel |
author_sort | Santos-Barriopedro, Irene |
collection | PubMed |
description | Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease. |
format | Online Article Text |
id | pubmed-8373943 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-83739432021-09-02 Off-the-shelf proximity biotinylation for interaction proteomics Santos-Barriopedro, Irene van Mierlo, Guido Vermeulen, Michiel Nat Commun Article Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease. Nature Publishing Group UK 2021-08-18 /pmc/articles/PMC8373943/ /pubmed/34408139 http://dx.doi.org/10.1038/s41467-021-25338-4 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Santos-Barriopedro, Irene van Mierlo, Guido Vermeulen, Michiel Off-the-shelf proximity biotinylation for interaction proteomics |
title | Off-the-shelf proximity biotinylation for interaction proteomics |
title_full | Off-the-shelf proximity biotinylation for interaction proteomics |
title_fullStr | Off-the-shelf proximity biotinylation for interaction proteomics |
title_full_unstemmed | Off-the-shelf proximity biotinylation for interaction proteomics |
title_short | Off-the-shelf proximity biotinylation for interaction proteomics |
title_sort | off-the-shelf proximity biotinylation for interaction proteomics |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373943/ https://www.ncbi.nlm.nih.gov/pubmed/34408139 http://dx.doi.org/10.1038/s41467-021-25338-4 |
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