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Systems-Wide Site-Specific Analysis of Glycoproteins

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Mass Spectrometry-Based Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2718))

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Abstract

Glycosylation is one of the most common and complex post-translation modifications that influence the structural and functional properties of proteins. Glycoproteins are highly heterogeneous and exhibit site- and protein-specific expression differences. Mass spectrometry in combination with liquid chromatography has emerged as the most powerful tool for the comprehensive characterization of glycosylation. The analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for their glycan heterogeneity at both protein- and site-specific levels. Nevertheless, intact glycopeptide characterization is challenging as elucidation of the glycan and peptide moieties requires specific sample preparation workflows that, combined with the tandem mass spectrometry approach, enable the identification of single glycopeptide species. In this chapter, we provide a detailed description of the methods that include procedures for (i) proteolytic digestion using specific proteases, (ii) optional glycopeptide enrichment using hydrophilic interaction liquid chromatography, (iii) nano-LC-MS/MS analysis of glycopeptides, and (iv) data analysis for identification of glycopeptides. Together, our workflow provides a framework for the system-wide site-specific analysis of N- and O-glycopeptides derived from complex biological or clinical samples.

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Abbreviations

ACN:

Acetonitrile

CAA:

Chloroacetamide

CID:

Collision-induced dissociation

ETD:

Electron transfer dissociation

ETHcD:

Electron-transfer/higher-energy collision dissociation

EtOH:

Ethanol

FA:

Formic acid

HCD:

Higher-energy collisional dissociation

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HILIC:

Hydrophilic interaction chromatography

LC:

Liquid chromatography

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

PTMs:

Posttranslational modifications

RT:

Room temperature

SP3:

Single-pot solid-phase-enhanced sample preparation

TFA:

Trifluoroacetic acid

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Acknowledgments

We thank the Max Planck Society for the funding support.

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Authors and Affiliations

  1. Max Planck Unit for the Science of Pathogens, Berlin, Germany

    Kathirvel Alagesan & Emmanuelle Charpentier

  2. Institute for Biology, Humboldt University, Berlin, Germany

    Emmanuelle Charpentier

Authors
  1. Kathirvel Alagesan
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  2. Emmanuelle Charpentier
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Correspondence to Kathirvel Alagesan .

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Editors and Affiliations

  1. VIB-UGent Center for Medical Biotechnology, Ghent, Belgium

    Kris Gevaert

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Alagesan, K., Charpentier, E. (2023). Systems-Wide Site-Specific Analysis of Glycoproteins. In: Gevaert, K. (eds) Mass Spectrometry-Based Proteomics. Methods in Molecular Biology, vol 2718. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3457-8_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3457-8_9

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