Solid-phase extraction of N-linked glycopeptides (original) (raw)

Nature Protocols volume 2, pages 334–339 (2007)Cite this article

Abstract

Protein glycosylation is a common post-translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. _N_-linked glycosylation is prevalent in proteins on the extracellular membrane, and many clinical biomarkers and therapeutic targets are glycoproteins. Here, we describe a protocol for solid-phase extraction of _N_-linked glycopeptides and subsequent identification of _N_-linked glycosylation sites (_N_-glycosites) by tandem mass spectrometry. The method oxidizes the carbohydrates in glycopeptides into aldehydes, which can be immobilized on a solid support. The _N_-linked glycopeptides are then optionally labeled with a stable isotope using deuterium-labeled succinic anhydride and the peptide moieties are released by peptide-_N_-glycosidase. In a single analysis, the method identifies hundreds of _N_-linked glycoproteins, the site(s) of _N_-linked glycosylation and the relative quantity of the identified glycopeptides.

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Article 23 June 2022

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Acknowledgements

This work was supported with federal funds from the National Cancer Institute, National Institutes of Health, by grant R21-CA-114852 (to H.Z.) and by Federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, under contract no. N01-HV-8179 (to R. A.).

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Authors and Affiliations

  1. Institute for Systems Biology, Seattle, 98103, Washington, USA
    Yuan Tian, Yong Zhou, Sarah Elliott & Hui Zhang
  2. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, 21287, Maryland, USA
    Yuan Tian & Hui Zhang
  3. Swiss Federal Institute of Technology (ETH) Zurich and Faculty of Natural Sciences, University of Zurich, Zurich, CH-8093, Switzerland
    Ruedi Aebersold

Authors

  1. Yuan Tian
  2. Yong Zhou
  3. Sarah Elliott
  4. Ruedi Aebersold
  5. Hui Zhang

Corresponding author

Correspondence toHui Zhang.

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The authors declare no competing financial interests.

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Tian, Y., Zhou, Y., Elliott, S. et al. Solid-phase extraction of _N_-linked glycopeptides.Nat Protoc 2, 334–339 (2007). https://doi.org/10.1038/nprot.2007.42

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