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Proximity labeling in mammalian cells with TurboID and split-TurboID

Nature Protocols volume 15, pages 3971–3999 (2020)Cite this article

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Abstract

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein–protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase–related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein–protein interactions or organelle–organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5–7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).

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Fig. 1: Proximity-dependent biotinylation catalyzed by TurboID and split-TurboID.
Fig. 2: Workflow for performing a TurboID proteomic experiment.
Fig. 3: Example data characterizing ERM-targeted TurboID.
Fig. 4: Proteomic data analysis using the ratiometric approach.
Fig. 5: Example experimental design and analysis using split-TurboID for proteomic mapping of ER–mitochondria contacts.

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Data availability

The data presented in this paper have been previously published, and associated raw data are provided in the original articles6,7.

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Acknowledgements

This work was supported by NIH R01-DK121409 (to A.Y.T. and S.A.C.) and the Stanford Wu Tsai Neurosciences Institute Big Ideas Initiative (to A.Y.T.). K.F.C. was supported by NIH Training Grant 2T32CA009302-41 and the Blavatnik Graduate Fellowship. T.C.B. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation (DRG-2391-20). A.Y.T. is an investigator of the Chan Zuckerberg Biohub.

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Author notes

  1. These authors contributed equally: Kelvin F. Cho, Tess C. Branon.

Authors and Affiliations

  1. Cancer Biology Program, Stanford University, Stanford, CA, USA

    Kelvin F. Cho

  2. Department of Genetics, Stanford University, Stanford, CA, USA

    Kelvin F. Cho & Alice Y. Ting

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Tess C. Branon

  4. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Namrata D. Udeshi, Samuel A. Myers & Steven A. Carr

  5. Department of Biology, Stanford University, Stanford, CA, USA

    Alice Y. Ting

  6. Department of Chemistry, Stanford University, Stanford, CA, USA

    Alice Y. Ting

  7. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Alice Y. Ting

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Contributions

K.F.C., T.C.B, N.D.U., S.A.M., S.A.C., and A.Y.T. contributed to the writing and editing of the manuscript.

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Correspondence to Alice Y. Ting.

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Competing interests

A.Y.T. and T.C.B. have filed a patent application covering some aspects of this work.

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Related links

Key references using this protocol:

Branon, T. C. et al. Nat. Biotechnol. 36, 880–887 (2018): https://www.nature.com/articles/nbt.4201

Cho, K. F. et al. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020): https://www.pnas.org/content/117/22/12143

Supplementary information

Reporting Summary

Supplementary Table 1

Human proteome of proteins, annotated by whether each protein was previously detected in a PL proteomic experiment from our lab (regions include: mitochondrial matrix6,15, mitochondrial intermembrane space76, mitochondrial nucleoid77, ER membrane6,7,78, outer mitochondrial membrane7,78, ER-mitochondria contact sites7,78, nucleus6, synaptic cleft20, and cytosol6,7,78). For each protein, the compartment(s) in which they were detected are listed.

Supplementary Table 2

Compilation of data from previous PL proteomic mapping experiments performed by our lab, categorized by organelle/region of interest (each tab is a different subcellular compartment). In each tab, the relevant studies and corresponding enrichment ratios (SILAC, TMT, or iTRAQ) for proteins detected above the respective cutoffs are provided. Data are included for the mitochondrial matrix6,15 (Tab 1), mitochondrial intermembrane space76 (Tab 2), mitochondrial nucleoid77 (Tab 3), ER membrane6,7,78 (Tab 4), outer mitochondrial membrane7,78 (Tab 5), ER-mitochondria contact sites7,78 (Tab 6), nucleus6 (Tab 7), synaptic cleft20 (Tab 8), and cytosol6,7,78 (Tab 9).

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Cho, K.F., Branon, T.C., Udeshi, N.D. et al. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nat Protoc 15, 3971–3999 (2020). https://doi.org/10.1038/s41596-020-0399-0

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