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Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site–specific stable isotope tagging

Nature Protocols volume 1, pages 3019–3027 (2006)Cite this article

Abstract

Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotope-coded glycosylation site–specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column–mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, 18O18O, specifically at the N-glycosylation site; and (4) identification of 18O-tagged peptides by liquid chromatography–coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.

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Figure 1: Strategy for large-scale identification of N-linked glycoproteins by IGOT-LC-MS/MS.
Figure 2: N-linked glycans and their binding lectins.
Figure 3: PNGase-mediated isotope-coded glycosylation site–specific stable isotope tagging (IGOT) in H218O.
Figure 4: Mass spectra of N-linked glycopeptides after IGOT.

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Acknowledgements

This work was supported in part by grants for the Structural Glycomics Project from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and for the Integrated Proteomics Project, Pioneer Research on Genome the Frontier from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by a grant-in-aid for Scientific Research from MEXT of Japan.

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Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji, 192-0397, Tokyo, Japan

    Hiroyuki Kaji, Yoshio Yamauchi & Toshiaki Isobe

  2. Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu, Tokyo, 183-8509, Japan

    Nobuhiro Takahashi

  3. CREST, Japan Science and Technology Agency, Honmachi 4-1-8, Kawaguchi-shi, 332-0012, Saitama, Japan

    Nobuhiro Takahashi & Toshiaki Isobe

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  1. Hiroyuki Kaji
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Correspondence to Toshiaki Isobe.

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Kaji, H., Yamauchi, Y., Takahashi, N. et al. Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site–specific stable isotope tagging. Nat Protoc 1, 3019–3027 (2006). https://doi.org/10.1038/nprot.2006.444

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