The Chromatography Forum

of Delaware Valley

HILIC – It’s not just for Glycans: Aiding the identification of Glycopeptides and Modified Peptides by Modeling HILIC Retention

Many of the chemical modifications that play a role in biological processes and protein aging often change the hydrophilicity of the modified amino acid’s side chain. These alterations can include the attachment of a single (or multiple) monosaccharides, oxidation of methionine, the deamidation of asparagine, and isomerization of aspartic acid, among others.   Using conventional reverse-phase approaches, the peptides with and without these modifications often do not exhibit sufficient separation from one another, or when these can be chromatographically resolved the shift in retention is easily predictable.  Changes in hydrophilicity directly alter a peptide’s retention on hydrophilic interaction liquid chromatography (HILIC) columns. 

We have created a HILIC model that can predict the retention of peptides with various modifications.  We have shown the ability to chromatographically resolve and quantitate products of deamidation, isomerization, oxidation, O-GlcNAcylation, and glycation.  We have derived retention coefficients for these modified amino acids, and have incorporated them into a model that predicts peptide retention on HILIC columns.

The analysis of an extensive range of procainamide tagged N-linked glycans has led to a HILIC retention model for this class of biomolecule.  The N-glycan model was combined with the peptide model, described above, to produce a prediction model for glycopeptides.  The efficacy of the combined model was evaluated by comparing experimental data obtained via LC-MS analysis of human serum IgGs to those calculated with the model. 

We feel that the unified chromatographic model (amino acids, modified amino acids, N-linked glycans) will facilitate the identification and quantification of peptides containing these modifications.