Optimization of proximity labeling in endothelial cells: overcoming endogenous biotin interference and cost barriers.

Abstract

Proximity labeling has become a powerful technique for mapping protein-protein interactions under physiologically relevant conditions, with TurboID offering high enzymatic activity and rapid labeling. However, its application in endothelial systems has been limited, partly due to the presence of endogenous biotin in specialized media, which reduces labeling specificity. Here, we optimized TurboID-mediated proximity labeling in brain microvascular endothelial cells by addressing two key challenges: endogenous biotin interference and cost-effective depletion. We discovered that endothelial cell medium contains substantial biotin levels, which saturate TurboID labeling and obscure the effects of exogenous biotin. Using High Capacity NeutrAvidin^™ agarose, we developed a simple and economical method to deplete endogenous biotin, reducing background biotinylation dramatically. We then defined the optimal condition for efficient labeling with minimal toxicity. Using TKS4, a scaffold protein critical for podosome formation, we validated this workflow in brain microvascular endothelial cells and confirmed the efficiency of streptavidin-based enrichment of biotinylated proteins. This study provides a validated and accessible TurboID workflow for endothelial cells, enabling more precise and cost-effective discovery of dynamic protein interaction networks relevant to vascular integrity and disease.