Abstract
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 clustered regularly interspaced short palindromic repeats (CRISPR)-based tagging, thus prohibiting the use of such assays in cell types that are difficult to transfect or transduce. We recently developed an ‘off-the-shelf’ proximity biotinylation method that 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. In principle, any scientist can perform this protocol within 3 days, although generating the proteomics data requires access to a high-end liquid chromatography–mass spectrometry setup. Data analysis and data visualization are relatively straightforward and can be performed using any type of software that converts raw mass spectrometry spectra files into identified and quantified proteins. The protocol has been optimized for nuclear targets but may also be adapted to other subcellular regions of interest.
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Data availability
The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE27 partner repository with the dataset identifier PXD030963, and processed data are provided in Supplementary Table 1. Source data are provided with this paper.
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Acknowledgements
We thank members of the Vermeulen lab for input during the development of the off-the-shelf proximity labeling approach. The Vermeulen lab is part of the Oncode Institute, which is partly funded by the Dutch Cancer Society. This work is further supported by an ERC consolidator grant to M.V. (771059). I.S.B. is supported by a Marie Sklodowska-Curie postdoctoral fellowship under the European Union’s Horizon 2020 research and innovation program (grant no. 835908). G.v.M. is supported by an EMBO long-term fellowship (no. 895-2020).
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I.S.B. and G.v.M. contributed equally to this work. I.S.B. and G.v.M. performed experiments and data analyses. I.S.B., G.v.M. and M.V. wrote the manuscript. All authors read and approved the final manuscript.
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I.S.B., G.v.M. and M.V. have filed a patent related to this work (PCT/NL2022/050197).
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Nature Protocols thanks Dalia Barsyte-Lovejoy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Santos-Barriopedro, I. et al. Nat. Commun. 12, 5015 (2021): https://doi.org/10.1038/s41467-021-25338-4
Supplementary information
Supplementary Table 1
Processed data related to MS measurements described in Figs. 2 and 3
Source data
Source Data Fig. 1
Unprocessed western blots
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Santos-Barriopedro, I., van Mierlo, G. & Vermeulen, M. Off-the-shelf proximity biotinylation using ProtA-TurboID. Nat Protoc 18, 36–57 (2023). https://doi.org/10.1038/s41596-022-00748-w
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DOI: https://doi.org/10.1038/s41596-022-00748-w
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