Dataset Information

‘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 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.

INSTRUMENT(S): Orbitrap Fusion, Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Primary Cell, Permanent Cell Line Cell, Cell Culture, Fibroblast

SUBMITTER: Irene Santos Barriopedro

LAB HEAD: Professor Michiel Vermeulen

PROVIDER: PXD025012 | Pride | 2021-07-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20200703_HFX1_SA_ISB_protAunfixed_H3K9me3.7z	Other
	20200703_HFX1_SA_ISB_protAunfixed_H3K9me3_1.raw	Raw
	20200703_HFX1_SA_ISB_protAunfixed_H3K9me3_2.raw	Raw
	20200703_HFX1_SA_ISB_protAunfixed_H3K9me3_3.raw	Raw
	20200703_HFX1_SA_ISB_protAunfixed_IgG1.raw	Raw

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Publications

Off-the-shelf proximity biotinylation for interaction proteomics.

Santos-Barriopedro Irene I van Mierlo Guido G Vermeulen Michiel M

Nature communications 20210818 1

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 lysa ...[more]

PMID: 34408139

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