Proximity labeling in mammalian cells with TurboID and split-TurboID

Autor: Kelvin F. Cho, Namrata D. Udeshi, Alice Y. Ting, Steven A. Carr, Tess C. Branon, Samuel A. Myers
Rok vydání: 2020
Předmět:
Zdroj: Nature Protocols. 15:3971-3999
ISSN: 1750-2799
1754-2189
DOI: 10.1038/s41596-020-0399-0
Popis: 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). This protocol describes proximity labeling approaches using TurboID and split-TurboID, which can be used for mapping protein–protein interactions and organelle proteomes in live mammalian cells with nanometer spatial resolution.
Databáze: OpenAIRE
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