LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from ARF6-KO and WT mice followed by label-free quantification (LFQ)

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 and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To better understand the role of Arf6 in BECs, in this experiment we have compared MACS sorted BECs from Arf6-KO and WT 3-months-old mice and found 140 and 172 proteins to be significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins showed mRNA processing to be among the most affected biological processes consistent with our findings on the aged BEC proteome.