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. 2016 Sep;27(3):98-104.
doi: 10.7171/jbt.16-2703-003. Epub 2016 Aug 3.

Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC)

Yining Huang  1 , Yongxin Nie  2 , Barry Boyes  3 , Ron Orlando  1
Affiliations

Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC)

Yining Huang et al. J Biomol Tech. 2016 Sep.

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 α2-3 and α2-6 linkages, while these glycans remain attached to peptides.

Keywords: Fetuin; HILIC; IgGs; Isomeric Separation.

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Figures

FIGURE 1.
FIGURE 1.
Behavior of tryptic glycopeptides of fetuin during RP chromatography and HILIC. A) The total ion chromatogram (TIC; blue trace) and EIC (inset graph: purple trace of GP15-Bi-2SA; pink trace of GP15-Tri-3SA; green trace of GP15-Tri-4SA) from the RP separation and (B) the TIC (blue trace) and EIC (inset graph: purple trace of GP15-Bi-2SA; pink trace of GP15-Tri-3SA; green trace of GP15-Tri-4SA) of HILIC separation of trypsin-digested fetuin.
FIGURE 2.
FIGURE 2.
Comparison of the separation obtained from released N-glycans and glycopeptides of fetuin by HILIC. A) HILIC separation of ProA-labeled, released N-glycans. B) HILIC separation of glycopeptides with the same peptide backbone.
FIGURE 3.
FIGURE 3.
The HILIC separation of major N-glycopeptides in IgG1, IgG2, and IgG4.
FIGURE 4.
FIGURE 4.
HILIC SRM analysis of human serum IgGs demonstrating the ability to resolve isomeric glycopeptide glycoforms.
See this image and copyright information in PMC

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