LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from 3-, 6-, 12-, and 18-months-old mice and Apoe-KO mice at 3 months followed by label-free quantification (LFQ)

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Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry based proteomics. In this experiment, first we have compared MACS sorted BECs across multiple time points between 3 and 18 months of age. Using unsupervised cluster analysis, we found a segregation of age-related protein dynamics with biological functions including a downregulation of vesicle-mediated transport. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe. Therefor in our next proteomics experiment we compared BECs from 3-months-old Apoe-KO and WT mice and found 111 and 103 proteins to be up- and downregulated, respectively. Comparing the BEC proteomic signature of young Apoe-KO mice with the signature of aged (18-months-old) WT mice we found a positive correlation suggesting an accelerating effect of Apoe deficiency on BEC aging.