Proximity Dependent Biotin Labelling in Zebrafish for Proteome and Interactome Profiling

Area of Science:

• Molecular Biology
• Proteomics
• Zebrafish Genetics

Background:

• Protein interaction networks are crucial for understanding biological processes.
• Conventional methods struggle with weak or transient protein interactions.
• Proximity-dependent biotinylation (BioID) and mass spectrometry (MS) are powerful tools for proteomic analysis.

Purpose of the Study:

• To apply engineered TurboID ligase for in vivo proteomic mapping and interactor screening in zebrafish.
• To develop and detail the Biotin Labelling In Tagged Zebrafish (BLITZ) method.
• To create novel transgenic zebrafish lines for TurboID-dGBP expression.

Main Methods:

• Generated transgenic zebrafish expressing TurboID fused to a GFP-binding nanobody (dGBP).
• Crossed TurboID-dGBP lines with existing GFP-tagged lines for targeted labeling.
• Incubated zebrafish embryos with biotin and analyzed biotinylated proteins via MS.

Main Results:

• Successfully established TurboID-dGBP zebrafish lines for proximity-dependent biotin labeling.
• Demonstrated the BLITZ method's efficacy in mapping local proteomes in live zebrafish.
• Enabled screening of novel protein interactors using this in vivo approach.

Conclusions:

• The BLITZ method provides a robust protocol for in vivo protein interaction mapping in zebrafish.
• TurboID-dGBP zebrafish lines facilitate efficient identification of protein interactors and local proteomes.
• This approach overcomes limitations of conventional methods for studying weak or transient interactions.