Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC)

Abstract

The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in a2-3 and a2-6 linkages, while these glycans remain attached to peptides.

Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder

“…It was found that both reverse phase and porous graphitized carbon (PGC) chromatography could not separate the individual components. Gratifyingly, hydrophilic interaction chromatography (HILIC) [22], a technique used to separate highly polar compounds [23], showed an incredible ability to fractionate SGP into homogeneous constituents (Fig. 3A).…”

Hydrophilic interaction liquid chromatography in the separation of glycopeptides and their isomers

“…Separation of these isomeric structures is important because changes in the abundance of these isomers can serve as a marker for monitoring of diseases [18]. Huang et al showed HILIC separation of isomeric N -glycan structures, such as sialylated N -glycan isomers differing in α2–3 and α 2–6 linkages, while these glycans remain attached to peptides [19]. Direct analysis of glycopeptides when glycans remain attached on peptides, which has the advantage that the glycosylation can be assigned to specific locations on the protein, is more difficult challenge than glycomic profiling [19–23].…”

LC‐MS/MS isomeric profiling of permethylated N‐glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients

“…LC‐MS/MS is currently considered a great tool for biomolecule identification, enabling simultaneous glycan isomeric separation and structural assignment. Various separation techniques have been developed to resolve glycan isomers at both the glycan and glycopeptide levels . Hydrophilic liquid interaction chromatography (HILIC) coupled with reducing end labeling or derivatization is an excellent approach to separate glycans or glycopeptides .…”