LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from Arf6-GFP-AAV vs GFP-AAV treated WT mice 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 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-GFP-AAV vs GFP-AAV treated 3-months-old WT mice and found 86 and 110 proteins to be significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed vesicle-mediated transport, activation of GTPase activity, and ER to Golgi vesicle-mediated transport to be among the most significantly affected biological processes.