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Nat Protoc DOI:10.1038/s41596-020-0399-0

Proximity labeling in mammalian cells with TurboID and split-TurboID.

Publication TypeJournal Article
Year of Publication2020
AuthorsCho, KF, Branon, TC, Udeshi, ND, Myers, SA, Carr, SA, Ting, AY
JournalNat Protoc
Date Published2020 Nov 02
ISSN1750-2799
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).

DOI10.1038/s41596-020-0399-0
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/33139955?dopt=Abstract

Alternate JournalNat Protoc
PubMed ID33139955
Grant ListDRG-2391-20 / / Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation) /