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

Abstract

The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO® penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer arm linked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via α2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.

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

“…Another chromatographic option for glycopeptide separation is hydrophilic interaction liquid chromatography (HILIC) [ 15 ]. HILIC is a powerful LC mode for the separation of polar analytes insufficiently retained in RP-LC [ 10 , 16 ]. Moreover, HILIC is also attractive in glycopeptides enrichment [ 17 , 18 ], and HILIC has been applied in the separation of the isomeric glycoforms differing only in linkage position and/or branching [ 15 , 19 , 20 , 21 ].…”

Analysis of site and structure specific core fucosylation in liver disease progression using exoglycosidase-assisted data-independent LC-MS/MS

“…The glycopeptides were further simplified by sequential treatment with exoglycosidases (α2-3,6,8,9 neuraminidase followed by α1-2 and α1-3,4 fucosidases) and were subsequently analyzed by LC-MS/MS without any further glycopeptide enrichment. We verified completeness of the exoglycosidase digests by nano HILIC separation of the hemopexin glycopeptide (Supplemental Figure 1) as described previously 36 . This assures that we quantify core fucosylated glycoforms of each peptide but allows comparison of fucosylation on glycoforms with different degree of branching (see below).…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…The same SWPAVGNCSSALR glycoforms were separated on Halo Penta‐HILIC. The separation window was much wider in Halo Penta‐HILIC than Halo HILIC, and the resolution between each peak was also better .…”