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Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans

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Abstract

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

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Abbreviations

AGP:

Alpha-1-acid glycoprotein

CE:

Capillary electrophoresis

ESI:

Electrospray ionization

F:

Deoxyhexose (fucose)

H:

Hexose

HILIC:

Hydrophilic interaction chromatography

HPAEC:

High performance anion exchange chromatography

IgG:

Immunoglobulin G

IVIG:

Intravenous immunoglobulin

LC:

Liquid chromatography

MALDI:

Matrix-assisted laser desorption ionization

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

N:

N-Acetylhexosamine

NR :

Reducing end reduced N-acetylhexosamine

PGC:

Porous graphitic carbon

RP:

Reversed-phase

S:

N-Acetyl neuraminic acid

TOF:

Time-of-flight

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Acknowledgements

The authors would like to acknowledge Lotta Andersson and Teija Kupari for excellent technical assistance. The authors also wish to thank Dr. Manfred Wuhrer and his group at Leiden University Medical Center for sharing their expertise in glycan analytics.

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  1. Leena Valmu

    Present address: Biova Ltd, Suursuontie 19, 00630, Helsinki, Finland

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  1. Finnish Red Cross Blood Service, Kivihaantie 7, 00310, Helsinki, Finland

    Ilja Ritamo, Jarkko Räbinä, Suvi Natunen & Leena Valmu

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Ritamo, I., Räbinä, J., Natunen, S. et al. Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans. Anal Bioanal Chem 405, 2469–2480 (2013). https://doi.org/10.1007/s00216-012-6680-5

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