|Publication Type||Journal Article|
|Year of Publication||2020|
|Authors||Cho, KF, Branon, TC, Udeshi, ND, Myers, SA, Carr, SA, Ting, AY|
|Date Published||2020 Nov 02|
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 Journal||Nat Protoc|
|Grant List||DRG-2391-20 / / Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation) /|