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

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. Protein-protein interactions are essential for controlling cellular processes, but many of these interactions are low-affinity, transient, or occur only under specific conditions, making them difficult to study. Proximity labeling is a method that overcomes this challenge by using engineered enzymes to “tag” proteins that are near a protein of interest inside living cells. In this study, we optimized a proximity labeling technique called TurboID for use in human brain endothelial cells, which are critical for blood-brain barrier function. A key challenge we discovered was that standard endothelial growth medium contains high levels of biotin, which interferes with the labeling process. To address this, we systematically compared different biotin-depletion strategies and identified a cost-effective method that made labeling both precise and affordable. We then tested different biotin concentrations and labeling times to define optimal conditions and confirmed that the system successfully captured proteins associated with TKS4, a scaffold protein important for endothelial remodeling. This optimized workflow enables researchers across biology, neuroscience, and vascular medicine to explore dynamic protein networks with greater accuracy and at lower cost. We optimized TurboID-based proximity labeling workflow for brain microvascular endothelial cells by comparing different streptavidin-based reagents for endogenous biotin depletion. We then compared biotin concentrations and labeling times to establish short-term labeling conditions. ARTICLE HIGHLIGHTSTurboID offers rapid labeling within minutes and high catalytic activity, but its application in endothelial systems has been hindered by endogenous biotin present in specialized media.Our study focuses on optimizing TurboID for human brain microvascular endothelial cells to investigate interactomes of TKS4, a scaffold protein essential for endothelial podosome formation and vascular remodeling.High Capacity NeutrAvidinTM agarose provides a practical and cost-effective solution for large-scale depletion, reducing resource demands compared to traditional magnetic beads.Among the first, to our knowledge, our group optimized the TurboID workflow in brain endothelial cells, enabling studies of podosome-related signaling and vascular biology.The protocol provides a foundation for future applications in primary endothelial cells, organoid models, and in vivo systems to explore disease-relevant vascular networks. TurboID offers rapid labeling within minutes and high catalytic activity, but its application in endothelial systems has been hindered by endogenous biotin present in specialized media. Our study focuses on optimizing TurboID for human brain microvascular endothelial cells to investigate interactomes of TKS4, a scaffold protein essential for endothelial podosome formation and vascular remodeling. High Capacity NeutrAvidinTM agarose provides a practical and cost-effective solution for large-scale depletion, reducing resource demands compared to traditional magnetic beads. Among the first, to our knowledge, our group optimized the TurboID workflow in brain endothelial cells, enabling studies of podosome-related signaling and vascular biology. The protocol provides a foundation for future applications in primary endothelial cells, organoid models, and in vivo systems to explore disease-relevant vascular networks.