SureCLIP and DrCLIP: Genetic manipulation-free, generalizable interactome mapping for cells and zebrafish

Area of Science:

• Molecular Biology
• Proteomics
• Biochemistry

Background:

• Unbiased interactome mapping is crucial for understanding cellular processes.
• Current methods like co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and proximity labeling have limitations.
• co-IP/MS suffers from low sensitivity and robustness due to non-covalent interactions and protein carryover, while proximity labeling requires genetic manipulation.

Purpose of the Study:

• To develop a robust and sensitive method for unbiased interactome mapping of endogenous proteins.
• To overcome the limitations of existing co-IP/MS and proximity labeling techniques.
• To establish a workflow applicable to both cell cultures and whole animal models.

Main Methods:

• Development of a reversible crosslinker-mediated co-IP/MS workflow.
• Introduction of SureCLIP (Subcellular-resolution, Reversible Crosslinker-mediated co-Immunoprecipitation) for cultured cells.
• Adaptation of SureCLIP into DrCLIP (Danio rerio adaptation of SureCLIP) for zebrafish larvae.

Main Results:

• The SureCLIP and DrCLIP workflows effectively map protein-protein interactions (PPIs) with high sensitivity.
• These methods minimize bait and immunoglobulin (IgG) co-elution, reducing experimental artifacts.
• SureCLIP and DrCLIP successfully identified novel interactors for focal adhesion and nuclear envelope proteins, and profiled the kindlin-2 interactome in zebrafish embryos without genetic manipulation.

Conclusions:

• SureCLIP and DrCLIP provide a powerful and generalizable platform for native interactome definition.
• These techniques enable unbiased PPI mapping in both 2D cell cultures and whole organisms.
• The methods offer improved sensitivity and robustness compared to existing approaches, facilitating the discovery of novel biological interactions.