Influence of different N- and O-linked carbohydrates on the retention times of synthetic peptides in reversed-phase high-performance liquid chromatography.

Glycopeptides consisting of 6-19 amino acid residues and different mono- and disaccharides attached to single asparagine and serine residues were synthesized on solid-phase and were characterized by reversed-phase high-performance liquid chromatography and circular dichroism. It was shown that the decreased retention times due to glycosylation could be correlated with the increasing length of the sugar moiety. Phosphorylation of the same sequences reduced the retention times 1.6 times more than glycosylation with monosaccharides did. The binding to the column was dependent on the structure of the disaccharide when derivatized and glycosylated asparagine, the building block of N-glycopeptide syntheses was studied. However, this structural dependence of the elution times disappeared in the final glycopeptides. Although both glycosylation and phosphorylation resulted in altered secondary structure of the peptide backbone, it appears that the retention times reflect the increased hydrophilicity more strongly than induced conformational orientation on the surface of the bonded phase.