Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the IGOT strategy.
Figure 2: Mass spectra of tryptic peptides of chicken ovomucoid obtained by the application of IGOT.
Figure 3: A portion of the MS/MS spectra of a glycopeptide identified by IGOT.
Figure 4: Mass spectra of the glycopeptide LNNGSLAFATVLK.

References

  1. Abbott, A. A post-genomic challenge: learning to read patterns of protein synthesis. Nature 402, 715–720 (1999).

    Article  CAS  Google Scholar 

  2. Oliver, S. Guilt-by-association goes global. Nature 403, 601–603 (2000).

    Article  CAS  Google Scholar 

  3. Kumar, A. & Snyder, M. Protein complexes take the bait. Nature 415, 123–124 (2002).

    Article  CAS  Google Scholar 

  4. MacCoss, M.J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. USA 99, 7900–7905 (2002).

    Article  CAS  Google Scholar 

  5. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  Google Scholar 

  6. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).

    Article  CAS  Google Scholar 

  7. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    Article  CAS  Google Scholar 

  8. Goshe, M.B. et al. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal. Chem. 73, 2578–2586 (2001).

    Article  CAS  Google Scholar 

  9. Goshe, M.B. et al. Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins. Anal. Chem. 74, 607–616 (2002).

    Article  CAS  Google Scholar 

  10. Geng, M., Zhang, X., Bina, M. & Regnier, F. Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests. J. Chromatogr. B Biomed. Sci. Appl. 752, 293–306 (2001).

    Article  CAS  Google Scholar 

  11. Xiong, L. & Regnier, F.E. Use of a lectin affinity selector in the search for unusual glycosylation in proteomics. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 782, 405–418 (2002).

    Article  CAS  Google Scholar 

  12. Gonzalez, J. et al. A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant α-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water. Anal. Biochem. 205, 151–158 (1992).

    Article  CAS  Google Scholar 

  13. Kuster, B. & Mann, M. 18O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching. Anal. Chem. 71, 1431–1440 (1999).

    Article  CAS  Google Scholar 

  14. Harvey, D.J., Wing, D.R., Kuster, B. & Wilson, I.B. Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins. J. Am. Soc. Mass Spec. 11, 564–571 (2000).

    Article  CAS  Google Scholar 

  15. Hirabayashi, J., Hayama, K., Kaji, H., Isobe, T. & Kasai, K. Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans. J. Biochem. (Tokyo) 132, 103–114 (2002).

    Article  CAS  Google Scholar 

  16. Altmann, F., Schweiszer, S. & Weber, C. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. Glycoconj. J. 12, 84–93 (1995).

    Article  CAS  Google Scholar 

  17. Bause, E. & Legler, G. The role of the hydroxy amino acid in the triplet sequence Asn-Xaa-Thr (Ser) for the N-glycosylation step during glycoprotein biosynthesis. Biochem. J. 195, 639–644 (1981).

    Article  CAS  Google Scholar 

  18. Han, D.K., Eng, J., Zhou, H. & Aebersold, R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946–951 (2001).

    Article  CAS  Google Scholar 

  19. Cagney, G. & Emili, A. De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging. Nat. Biotechnol. 20, 163–170 (2002).

    Article  CAS  Google Scholar 

  20. Stenico, M., Lloyd, A.T. & Sharp, P.M. Codon usage in Caenorhapditis elegans: delineation of translational selection and mutational biases. Nucleic Acids Res. 22, 2437–2446 (1994).

    Article  CAS  Google Scholar 

  21. Mawuenyega, G.K. et al. Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography–tandem mass spectrometry. J. Proteome Res. 2, 23–35 (2002).

    Article  Google Scholar 

  22. Isobe, T., Yamauchi, Y., Taoka, M. & Takahashi, N. in Proteins and Proteomics: A Laboratory Manual (ed. Simpson, R.J.) 869–876 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003).

    Google Scholar 

  23. Natsume, T., et al. A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal. Chem. 74, 4725–4733 (2002).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Toshiaki Isobe.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt829

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing