Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins (original) (raw)

Nature Biotechnology volume 21, pages 667–672 (2003)Cite this article

Abstract

We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column–mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase–mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography–mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.

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Figure 1: Schematic representation of the IGOT strategy.

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Figure 2: Mass spectra of tryptic peptides of chicken ovomucoid obtained by the application of IGOT.

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Figure 3: A portion of the MS/MS spectra of a glycopeptide identified by IGOT.

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Figure 4: Mass spectra of the glycopeptide LNNGSLAFATVLK.

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Acknowledgements

This work was supported in part by grants for the Integrated Proteomics System Project, Pioneer Research on Genome the Frontier from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji, 192-0397, Tokyo, Japan
    Hiroyuki Kaji, Haruna Saito, Takashi Shinkawa, Masato Taoka & Toshiaki Isobe
  2. The Integrated Proteomics System Project, Pioneer Research on Genome the Frontier, MEXT, c/o Tokyo Metropolitan University, Hachioji, 192-0397, Tokyo, Japan
    Yoshio Yamauchi, Nobuhiro Takahashi & Toshiaki Isobe
  3. Department of Biological Chemistry, Faculty of Pharmaceutical Science, Teikyo University, Sagamiko, 199-0195, Kanagawa, Japan
    Jun Hirabayashi & Ken-ichi Kasai
  4. Department of Applied Biological Science, and Department of Biotechnology, United Graduate School of Agriculture, Tokyo University of Agriculture & Technology, Fuchu, 183-8509, Tokyo, Japan
    Nobuhiro Takahashi
  5. Proteomics Division, Medical Research Institute, The University of Tokyo, Minato-ku, 108-8639, Tokyo, Japan
    Toshiaki Isobe

Authors

  1. Hiroyuki Kaji
  2. Haruna Saito
  3. Yoshio Yamauchi
  4. Takashi Shinkawa
  5. Masato Taoka
  6. Jun Hirabayashi
  7. Ken-ichi Kasai
  8. Nobuhiro Takahashi
  9. Toshiaki Isobe

Corresponding author

Correspondence toToshiaki Isobe.

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The authors declare no competing financial interests.

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Kaji, H., Saito, H., Yamauchi, Y. et al. Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.Nat Biotechnol 21, 667–672 (2003). https://doi.org/10.1038/nbt829

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