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Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging

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Abstract

The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification.

Detection of biotinylated O-GlcNAz proteins in MCF-7 cells

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Acknowledgements

The authors are grateful to Sébastien Vidal, Philippe Belmont and Tony Lefebvre for helpful discussions, to Peter Goekjian for critical reading of the manuscript and to Adeline Page for nano-HPLC-MS/MS experiments.

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Authors and Affiliations

  1. UMR CNRS 5180, Sciences Analytiques, Université de Lyon, Lyon 1, 43 Bld du 11 novembre 1918, 69622, Villeurbanne cedex, France

    Caroline Gurcel, Catherine Fonbonne, Arnaud Salvador & Jérôme Lemoine

  2. UMR USTL/CNRS 8576, Unité de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, 59655, Villeneuve d’Ascq cedex, France

    Anne-Sophie Vercoutter-Edouart, Marlène Mortuaire & Jean-Claude Michalski

Authors
  1. Caroline Gurcel
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  2. Anne-Sophie Vercoutter-Edouart
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  3. Catherine Fonbonne
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  4. Marlène Mortuaire
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  5. Arnaud Salvador
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  6. Jean-Claude Michalski
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  7. Jérôme Lemoine
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Corresponding author

Correspondence to Jérôme Lemoine.

Additional information

Caroline Gurcel and Anne-Sophie Vercoutter-Edouart contributed equally to this work.

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Gurcel, C., Vercoutter-Edouart, AS., Fonbonne, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). https://doi.org/10.1007/s00216-008-1950-y

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  • DOI: https://doi.org/10.1007/s00216-008-1950-y

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