A fishing technology of glycated peptides

Glycation is a non-enzymatic, irreversible reaction between glucose and amino functions of proteins at the N-terminus and/or Lys or Arg side-chains. Amadori products are well known in food chemistry, but also as intermediates of Advanced Glycation End Products (AGEs) involved in complications of Diabetes and other neurodegenerative diseases [1]. The non-enzymatic glycation of peptides and proteins in human beings occurs spontaneously in vivo when the concentration of glucose is highly significant in blood: the sugar-modified peptides change their functional conformation and lose their normal activity. Thus, detection and sequencing of modified peptides is crucial. Solid supports modified with a phenyl boronate moiety can be useful tools to fish out peptide-sugar conjugates in biological fluids. In our previous work, ChemMatrix® Rink (CMRR) resin with a lysine containing two phenyl boronate (PhB) units, PhB-Lys(PhB)-CMRR, resin was found to be able to capture only the peptides containing deoxyfructosyl-lysine moieties, which can be efficiently and specifically detected by MS experiments [2]. In order to optimize the "fishing" methodology, in terms of the efficiency and selectivity, we compared the capture to deoxyfructosylated peptides by (i) two different resins: ChemMatrix® Rink resin (CMRR) and ω-Aminohexyl-Agarose (ω-AHA) and by (ii) the same resin, CMRR and ω-AHA, containing one and/or two phenylboronic acid moieties. To compare the different capture properties of both resins, the ω-AHA was functionalized on the amino function with Rink Linker present in the commercially available ChemMatrix® Rink resin. Therefore, we obtained the new solid support Agarose Rink Resin (AGRR). Different synthesized solid supports were tested with different Amadori peptides vs. non-Amadori peptides and their mixtures. The obtained results suggest that the type of solid support does not have an effect on binding efficiency, but only on the selectivity. Moreover, the level of substitution of the phenylboronic acid ring, mono-substituted, performed better than bi-substituted.