Blood Brain Barrier Proteomics

The identification of proteins that are differentially expressed in the organ microvasculature has the potential to address a variety of problems ranging from the analysis of disease pathogenesis to drug targeting for particular tissues. Oftentimes, the gene expression levels do not correlate directly with the level of protein expression seen in the cells. Thus, genomics studies cannot tell the entire story. This figure describes a complimentary methodology designed to analyze differential protein expression in the brain microvasculature, that could be applied to other organs, in order to identify proteins that are uniquely expressed in a given tissue. The technology merges a tissue-specific polyclonal antiserum with a cDNA library expression cloning system. The tissue-specific antiserum is subtracted with protein extracts from control tissues to remove those antibodies that recognize common antigenic proteins. Then, the depleted antiserum is used to expression clone tissue-specific proteins from a cDNA library expressed in mammalian cells.

The methodology was evaluated with a rabbit polyclonal antiserum prepared against purified bovine brain capillaries. The antiserum was absorbed with acetone powders of liver and kidney and then used to screen a bovine brain capillary cDNA library in COS cells (mammalian cells). The initial clones detected with this expression methodology were the Lutheran membrane glycoprotein and CD46. Both of these proteins have BBB-enriched expression and are glycosylated membrane proteins. This subtractive expression cloning methodology provides a new approach to 'vascular proteomics', and to the detection of proteins specifically expressed at the brain microvasculature.