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Off-the-shelf proximity biotinylation for interaction proteomics.

ABSTRACT: 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.

SUBMITTER: Santos-Barriopedro I

PROVIDER: S-EPMC8373943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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‘Off the shelf’ proximity biotinylation for interaction proteomics

Project 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 of interest using an endogenous antibody against the bait protein. 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, the histone modification H3K9me3 and the chromatin remodeler BRG1. Amongst the main findings, our experiments revealed Flywch1 as an essential protein that interacts with H3K9me3-marked centromeric heterochromatin. 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.

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Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches.

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2022-10-17 | PXD030963 | Pride

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