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

Hydrophilic interaction liquid chromatography in the separation of glycopeptides and their isomers 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 separa- tion of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification. . . . . Keywords Glycoproteomics Glycopeptides Hemopexin Hydrophilic interaction http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Analytical and Bioanalytical Chemistry Springer Journals

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

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature
Subject
Chemistry; Analytical Chemistry; Biochemistry, general; Laboratory Medicine; Characterization and Evaluation of Materials; Food Science; Monitoring/Environmental Analysis
ISSN
1618-2642
eISSN
1618-2650
D.O.I.
10.1007/s00216-018-1150-3
Publisher site
See Article on Publisher Site

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 separa- tion of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification. . . . . Keywords Glycoproteomics Glycopeptides Hemopexin Hydrophilic interaction

Journal

Analytical and Bioanalytical ChemistrySpringer Journals

Published: May 28, 2018

References

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