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Comparative Study
. 2020 Oct 13;25(20):4655.
doi: 10.3390/molecules25204655.

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

Katarina Molnarova  1 , Petr Kozlík  1
Affiliations
Comparative Study

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

Katarina Molnarova et al. Molecules. .

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.

Keywords: glycopeptide separation; glycopeptides; glycoproteomics; hydrophilic interaction liquid chromatography; separation of glycopeptide isomers.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Separation of glycopeptides of hemopexin in HALO® penta- hydrophilic interaction liquid chromatography (HILIC) (A), Glycan ethylene bridged hybrid (BEH) amide (B), and ZIC-HILIC (C) columns. PEP1 refers to SWPAVGN187CSSALR, and PEP2 to ALPQPQN453VTSLLGCTH.
Figure 2
Figure 2
Separation of glycopeptides of IgG in HALO® penta-HILIC (A), Glycan BEH amide (B), and ZIC-HILIC (C) columns. PEP1 refers to EEQYN180STYR and PEP2 to EEQFN176STFR.
Figure 3
Figure 3
Normalized EIC chromatograms of A2G2F1 glycoforms of SWPAVGN187CSSALR (A–C) and ALPQPQN453VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to SWPAVGN187CSSALR, and PEP2 to ALPQPQN453VTSLLGCTH.
Figure 4
Figure 4
Normalized EIC chromatograms of A2G2S1 glycoforms of SWPAVGN187CSSALR (A–C) and ALPQPQN453VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to SWPAVGN187CSSALR, and PEP2 to ALPQPQN453VTSLLGCTH.
See this image and copyright information in PMC

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