A TurboID-Based Protocol for Efficient Interacting Proteins Identification Using Proximity Tagging Technology

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Protein-protein interactions (PPIs) are crucial for cellular signaling and biological processes.
• Conventional PPI identification methods like yeast two-hybrid and co-immunoprecipitation have limitations in spatiotemporal resolution and detecting transient interactions.
• Proximity-dependent biotinylation (PDB) coupled with mass spectrometry (MS) offers a powerful approach to map the spatial proteome.

Purpose of the Study:

• To provide a detailed protocol for TurboID-based proximity labeling.
• To identify potential interacting proteins of a target protein of interest.
• To leverage the high catalytic efficiency of TurboID for rapid protein interaction mapping.

Main Methods:

• Utilizing TurboID, an engineered biotin ligase with high catalytic efficiency.
• Performing proximity labeling experiments in living cells.
• Employing mass spectrometry (MS) for the identification of biotinylated proteins.

Main Results:

• TurboID enables rapid biotinylation of proximal proteins within minutes.
• The protocol facilitates the identification of a target protein's interacting partners.
• This technique enhances the mapping of the spatial proteome.

Conclusions:

• TurboID-based proximity labeling is an efficient method for mapping protein-protein interactions.
• This protocol overcomes limitations of traditional PPI identification techniques.
• The method is valuable for studying dynamic cellular processes and signaling pathways.