Luminal Cerebrovascular Proteomics.

Brain endothelial cells, which constitute the cerebrovasculature, form the first interface between the blood and brain and play essential roles in maintaining central nervous system (CNS) homeostasis. These cells exhibit strong apicobasal polarity, with distinct luminal and abluminal membrane compositions that crucially mediate compartmentalized functions of the vasculature. Existing transcriptomic and proteomic profiling techniques often lack the spatial resolution to discriminate between these membrane compartments, limiting insights into their distinct molecular compositions and functions. To overcome these limitations, we developed an in vivo proteomic strategy to selectively label and enrich luminal cerebrovascular proteins. In this approach, we perfuse a membrane-impermeable biotinylation reagent into the vasculature to covalently tag cell surface proteins exposed on the luminal side. This is followed by microvessel isolation and streptavidin-based enrichment of biotinylated proteins for downstream mass spectrometry analysis. Using this method, we robustly identified over 1,000 luminally localized proteins via standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques, achieving substantially improved enrichment of canonical luminal markers compared with conventional vascular proteomic approaches. Our method enables the generation of a high-confidence, compartment-resolved atlas of the luminal cerebrovascular proteome and offers a scalable platform for investigating endothelial surface biology in both healthy and disease contexts. Key features • Enables high-resolution proteomic profiling of the luminal surface of the brain vasculature in vivo. • Improves signal-to-noise ratio through an added microvessel isolation step, reducing nonspecific background. • Applied to uncover aging-related changes in luminal endothelial surface protein composition. • Adaptable for identifying therapeutic targets, transporters, signaling pathways, and disease-associated alterations in the luminal vascular environment across diverse biological contexts.