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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 12

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).


Alternate JournalNat Protoc
PubMed ID33139955
Grant ListR01 DK121409 / DK / NIDDK NIH HHS / United States
T32 CA009302 / CA / NCI NIH HHS / United States