Limitations of boronate affinity chromatography for the specific enrichment of fructose-derived early glycation products in protein analytics

Protein glycation, or non-enzymatic glycosylation, refers to the reaction of reducing sugars with amino groups in proteins to form Amadori and Heyns products for aldoses (e.g., glucose) and ketoses (e.g., fructose), respectively. While Amadori peptides have been well studied after enrichment by boronate affinity chromatography (BAC), it is often assumed that BAC also enriches the isomeric Heyns peptides, although the binding of Heyns rearrangement products seems unlikely due to the very low content of 1,2- and 1,3-cis-diols in their dominant tautomeric forms. For seven different tryptic peptide sequences derived from human plasma digests, we showed that the synthetic glucose-modified Amadori peptides can be enriched by BAC with high recovery rates, while the corresponding fructose-modified Heyns peptides did not bind, independent of the buffers and pH used. Reduction of the carbonyl groups with borohydride, yielding the corresponding hexitol-modified peptides, allowed enrichment of both the former Amadori and, more importantly, Heyns peptides.

Graphical Abstract

Molecular interactions of boronic acids with unreduced and reduced Amadori (ARPs) and Heyns rearrangement products (HRPs)