Off-the-shelf proximity labeling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE169317 | GEO | 2021/07/09
REPOSITORIES: GEO
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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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Project description:Off-the-shelf proximity labeling
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Project description:Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method
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Project description:Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding of post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tehers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, verified the co-localization of these identified proteins with baits proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient tool to identify proteins that are proximal to modified histones.
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