Discovery of Blood-Brain Barrier Transport-Related Surface Proteins by DIA-Orbitrap High-Resolution Mass Spectrometry

As a selective permeability interface, the Blood-Brain Barrier (BBB) restricts drug access to the brain, complicating theranostic strategies for neurological disorders. The selective permeability of the blood-brain barrier is primarily controlled by brain microvascular endothelial cells (BMECs). Cell surface proteins can be endocytosed into brain microvascular endothelial cells, serving as promising candidates for receptor-mediated transcytosis across the blood-brain barrier. However, to date, systematic analysis of the internalization of surface proteins in human blood-brain barrier models still has some limitations. In this study, we employed high-resolution mass spectrometry (HRMS) based on Data-Independent Acquisition (DIA) combined with cell surface biotinylation to comprehensively identify internalized surface proteins in human brain microvascular endothelial cells (HBEC-5i). This approach overcomes traditional proteomics limitations, enabling high-throughput quantification of hundreds of surface proteins. Our analysis identified 232 internalized cell-surface proteins, with 54 showing HBEC-5i specificity compared to dermal (HMEC-1) and pulmonary (HULEC-5a) microvascular endothelial cells. Through integration with mRNA expression data from public databases and immunohistochemical evidence from the Human Protein Atlas, we identified Plastin 3 (PLS3) and Collectin Subfamily Member 12 (COLEC12) as key candidate proteins. Both proteins exhibited elevated expression and distinct plasma membrane localization patterns in HBEC-5i cells. Our study highlights the unique advantages of DIA high-resolution mass spectrometry in profiling BBB-related proteins and provides valuable insights for developing targeted strategies to enhance central nervous system drug delivery.