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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.
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