Amino acid specificity of glycation and protein-AGE crosslinking reactivities determined with a dipeptide SPOT library

Advanced glycation end products (AGEs) contribute to changes in protein con formation, loss of function, and irreversible crosslinking. Using a library of dipeptides on cellulose membranes (SPOT library), we have developed an approach to systematically assay the relative reactivities of amino acid si de chains and the N-terminal amino group to sugars and protein-AGEs. The su gars react preferentially with cysteine or tryptophan when both the alpha-a mino group and the side chains are free. In peptides with blocked N-terminu s and free side chains, cysteine, lysine, and histidine were preferred. Cro sslinking of protein-AGEs to dipeptides with free side chains and blocked N termini occurred preferentially to arginine and tryptophan. Dipeptide SPOT libraries are excellent tools for comparing individual reactivities of ami no acids for nonenzymatic modifications, and could be extended to other che mically reactive molecules.