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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). Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your

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SIMILAR CONTENT BEING VIEWED BY OTHERS OFF-THE-SHELF PROXIMITY BIOTINYLATION USING PROTA-TURBOID Article 12 October 2022 OFF-THE-SHELF PROXIMITY BIOTINYLATION FOR INTERACTION PROTEOMICS

Article Open access 18 August 2021 ENGINEERING OF ULTRAID, A COMPACT AND HYPERACTIVE ENZYME FOR PROXIMITY-DEPENDENT BIOTINYLATION IN LIVING CELLS Article Open access 04 July 2022 DATA

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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. AUTHOR INFORMATION Author notes * These authors contributed equally: Kelvin F. Cho, Tess C. Branon. AUTHORS AND AFFILIATIONS * Cancer Biology

Program, Stanford University, Stanford, CA, USA Kelvin F. Cho * Department of Genetics, Stanford University, Stanford, CA, USA Kelvin F. Cho & Alice Y. Ting * Department of Molecular and

Cell Biology, University of California, Berkeley, Berkeley, CA, USA Tess C. Branon * Broad Institute of MIT and Harvard, Cambridge, MA, USA Namrata D. Udeshi, Samuel A. Myers & Steven

A. Carr * Department of Biology, Stanford University, Stanford, CA, USA Alice Y. Ting * Department of Chemistry, Stanford University, Stanford, CA, USA Alice Y. Ting * Chan Zuckerberg

Biohub, San Francisco, CA, USA Alice Y. Ting Authors * Kelvin F. Cho View author publications You can also search for this author inPubMed Google Scholar * Tess C. Branon View author

publications You can also search for this author inPubMed Google Scholar * Namrata D. Udeshi View author publications You can also search for this author inPubMed Google Scholar * Samuel A.

Myers View author publications You can also search for this author inPubMed Google Scholar * Steven A. Carr View author publications You can also search for this author inPubMed Google

Scholar * Alice Y. Ting View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Alice Y. Ting. ETHICS DECLARATIONS COMPETING INTERESTS A.Y.T. and T.C.B. have filed a patent application

covering some aspects of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. 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). RIGHTS

AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 17 May 2020 * Accepted: 18 August 2020 * Published: 02 November 2020

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