Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion

Mancera-Arteu, Montserrat; Giménez, Estela; Barbosa, J.; Sanz‐Nebot, Victoria

doi:10.1016/j.aca.2016.07.043

Cited by 44 publications

(59 citation statements)

References 35 publications

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“…The differentiation between both linkages is highly relevant since α2,3 and α2,6 linkages may have significantly different biological effects 29 . CE is not the only separation platform that enables separation of sialylated isomers: zwitterionic -HILIC and porous graphitized carbon (PGC)-LC have proven to be suitable for separation of glycan species with isomeric sialic acid linkages 34, 35 . Specific fragmentation features have been reported for anionic glycans, enabling the distinction between isomers 36, 37 .…”

Section: Resultsmentioning

confidence: 99%

Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis – electrospray ionization – mass spectrometry

Kammeijer

Jansen

Köhler

et al. 2017

Sci Rep

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Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis – mass spectrometry (CE–MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pKa values. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10−2 in pKa unit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.

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Section: Resultsmentioning

confidence: 99%

Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis – electrospray ionization – mass spectrometry

Kammeijer

Jansen

Köhler

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Similarly, HILIC and anion‐exchange chromatography have also been incorporated into a two‐dimensional on‐line LC system to separate isomeric sialylated N ‐glycans . In addition, HILIC in combination with exoglycosidase digestion can be used for the characterization and quantitative analysis of isomeric sialoglycans . Reversed phase chromatography (RPC) coupling a mobile phase containing ion‐pairing reagent has also been employed for the separation of sialoglycans derivatized by reductive amination .…”

Section: Oligosaccharidesmentioning

confidence: 99%

Mass spectrometry for protein sialoglycosylation

Zhang

Wang

et al. 2017

Mass Spectrometry Reviews

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Sialic acids are a family of structurally unique and negatively charged nine-carbon sugars, normally found at the terminal positions of glycan chains on glycoproteins and glycolipids. The glycosylation of proteins is a universal post-translational modification in eukaryotic species and regulates essential biological functions, in which the most common sialic acid is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid) (Neu5NAc). Because of the properties of sialic acids under general mass spectrometry (MS) conditions, such as instability, ionization discrimination, and mixed adducts, the use of MS in the analysis of protein sialoglycosylation is still challenging. The present review is focused on the application of MS related methodologies to the study of both N- and O-linked sialoglycans. We reviewed MS-based strategies for characterizing sialylation by analyzing intact glycoproteins, proteolytic digested glycopeptides, and released glycans. The review concludes with future perspectives in the field.

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“…HILIC is suitable for the analysis of polar analytes which are inadequately retained in RP-LC [1]. HILIC was successfully applied to glycopeptide enrichment [7,8], released glycan analysis [9,10], and separation of different glycoforms of peptides [11]. Although the directed analysis of glycoproteins provides site-specific information on glycans, the common approach to characterize glycans in HILIC is their enzymatic or chemical releas LC/MS analysis [12].…”

Section: Introductionmentioning

confidence: 99%

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

Kozlík

Šanda

Goldman

2017

Journal of Chromatography A

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Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.

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Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion

Cited by 44 publications

References 35 publications

Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis – electrospray ionization – mass spectrometry

Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis – electrospray ionization – mass spectrometry

Mass spectrometry for protein sialoglycosylation

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

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