Comparison of Different HILIC Stationary Phases in the Separation of Hemopexin and Immunoglobulin G Glycopeptides and Their Isomers.

Katarina Molnarova,Petr Kozlík

doi:10.3390/molecules25204655

Abstract

Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

Highlights

Glycosylation is one of the most important and extensive post-translation protein modifications [1].Protein glycosylation includes a complex series of enzymatic steps that result in the formation of protein-bound oligosaccharides with various biological functions [1,2,3]
We showed the effect of the composition of the glycans on the retention and separation of glycopeptides
Sialic acid in a glycan moiety significantly prolonged the retention times of hemopexin glycopeptides in the HALO® penta-hydrophilic interaction liquid chromatography (HILIC) column

Summary

Introduction

Glycosylation is one of the most important and extensive post-translation protein modifications [1].Protein glycosylation includes a complex series of enzymatic steps that result in the formation of protein-bound oligosaccharides with various biological functions [1,2,3]. Alterations in glycosylation patterns affect many biological processes, such as protein folding, intracellular sorting, secretion, and host-microbial recognition [4,5]. Abnormal protein glycosylation has been used as a diagnostic tool for many cancers, including breast, prostate, and ovarian cancer [6,7,8,9]. An alternative chromatographic mode to RP-LC in glycopeptide analysis is to use porous graphitized carbon (PGC). PGC has been successfully applied in the isomeric separation of different N-glycopeptides [13,14]. Another chromatographic option for glycopeptide separation is hydrophilic interaction liquid chromatography (HILIC) [15]. HILIC is attractive in glycopeptides enrichment [17,18], and HILIC

Methods

Results

Conclusion