Radical diffusion, not lifetime, determines the range of peroxidase-based proximity labelling

Area of Science:

• Cell Biology
• Proteomics
• Biochemistry

Background:

• Proximity labeling is vital for mapping protein interactions.
• Peroxidase-based methods (APEX2, HRP) are commonly used for biotinylation.
• Accurate spatial mapping is essential for understanding cellular organization.

Purpose of the Study:

• To analyze apical, lateral, and basal membrane proteomes in Drosophila follicular epithelium.
• To investigate the spatial fidelity of APEX2- and HRP-mediated proximity labeling.
• To identify factors influencing labeling radius and optimize reaction conditions.

Main Methods:

• APEX2- and HRP-mediated biotinylation in Drosophila follicular epithelium.
• Utilized Cadherin99c, Fasciclin 3, and Nidogen as bait proteins.
• Systematically varied biotin-phenol and biotin-SS-tyramide concentrations and reaction times.

Main Results:

• Standard peroxidase labeling conditions resulted in unexpected basal biotinylation signals, irrespective of bait localization.
• Long-range diffusion of phenoxy radicals (microns) was observed, exceeding the expected labeling radius.
• Basement membrane acted as a radical sink due to electron-rich amino acids.
• Optimized conditions (titrated reagents, shorter times) restored spatially faithful labeling.

Conclusions:

• Peroxidase-based proximity labeling's effective labeling distance is not limited by radical lifespan but by radical diffusion.
• Radical diffusion can lead to misleading spatial signatures if not carefully controlled.
• Optimization of reagent concentrations and reaction times is critical for accurate proximity labeling in cells and tissues.