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.

  • Protocol
  • Published:

Large-scale site-specific mapping of the O-GalNAc glycoproteome

Nature Protocols volume 15, pages 2589–2610 (2020)Cite this article

Subjects

Abstract

Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome is a crucial step toward understanding the biological significance of the modification. However, lack of suitable methodology, absence of consensus sequon of O-GalNAcylation sites and complex O-GalNAc glycan structures pose analytical challenges. We recently developed a mass spectrometry-based method called extraction of O-linked glycopeptides (EXoO) that enables large-scale mapping of site-specific mucin-type O-GalNAcylation sites. Here we provide a detailed protocol for EXoO, which includes seven stages of: (1) extraction and proteolytic digestion of proteins to peptides, (2) sequential guanidination and de-salting of peptides, (3) enrichment of glycopeptides, (4) solid-phase peptide conjugation and release of O-GalNAc glycopeptides using the OpeRATOR protease, (5) liquid chromatography with tandem mass spectrometry analysis of O-GalNAc glycopeptides, (6) identification of O-GalNAc glycopeptides by database search and (7) quantification of O-GalNAc glycopeptides. Using this protocol, thousands of O-GalNAcylation sites from hundreds of glycoproteins with information regarding site-specific O-GalNAc glycan can be identified and quantified from complex samples. The protocol can be performed by a researcher with basic proteomics skills and takes about 4 d to complete.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Workflow diagram of EXoO protocol and O-GalNAcylation pathway for biosynthesis of core 1 and 3 O-glycan structures.
Fig. 2: Reproducibility of the EXoO protocol.
Fig. 3: Quantitative performance of the EXoO protocol.
Fig. 4: MS spectra for the determination of OpeRATOR glycan specificity.
Fig. 5: MS2 spectra of O-GalNAc glycopeptides generated using EXoO.

Similar content being viewed by others

Data availability

The data are available in the PRIDE partner repository48 under the project identifier PXD009476.

Code availability

Extract_oxonium_ion_spectra_intensity.exe script is freely available at GitHub (download at https://github.com/appleTree2017/Extraction-of-O-linked-glycopeptides-EXoO-).

References

  1. Gahmberg, C. G. & Tolvanen, M. Why mammalian cell surface proteins are glycoproteins. Trends Biochem. Sci. 21, 308–311 (1996).

    CAS  PubMed  Google Scholar 

  2. Jang, J. H. & Hanash, S. Profiling of the cell surface proteome. Proteomics 3, 1947–1954 (2003).

    CAS  PubMed  Google Scholar 

  3. Kovtun, Y. V. & Goldmacher, V. S. Cell killing by antibody–drug conjugates. Cancer Lett. 255, 232–240 (2007).

    CAS  PubMed  Google Scholar 

  4. Selvaraj, P. et al. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Nature 326, 400–403 (1987).

    CAS  PubMed  Google Scholar 

  5. Orentas, R. J. et al. Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers. Front. Oncol. 2, 194 (2012).

    PubMed  PubMed Central  Google Scholar 

  6. Posey, A. D. Jr. et al. Engineered CAR T cells targeting the cancer-associated Tn-glycoform of the membrane mucin MUC1 control adenocarcinoma. Immunity 44, 1444–1454 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Westerlund, B. & Korhonen, T. K. Bacterial proteins binding to the mammalian extracellular matrix. Mol. Microbiol. 9, 687–694 (1993).

    CAS  PubMed  Google Scholar 

  8. Maddon, P. J. et al. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47, 333–348 (1986).

    CAS  PubMed  Google Scholar 

  9. Kennedy, S. Proteomic profiling from human samples: the body fluid alternative. Toxicol. Lett. 120, 379–384 (2001).

    CAS  PubMed  Google Scholar 

  10. Brockhausen, I. & Stanley, P. O-GalNAc glycans. in Essentials of Glycobiology 3rd edn (eds A. Varki et al.) (Cold Spring Harbor Laboratory Press, 2015).

  11. Tarelli, E., Smith, A. C., Hendry, B. M., Challacombe, S. J. & Pouria, S. Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. Carbohydr. Res. 339, 2329–2335 (2004).

    CAS  PubMed  Google Scholar 

  12. Arnold, J. N. et al. The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin. J. Immunol. 173, 6831–6840 (2004).

    CAS  PubMed  Google Scholar 

  13. Sasaki, H., Bothner, B., Dell, A. & Fukuda, M. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem. 262, 12059–12076 (1987).

    CAS  PubMed  Google Scholar 

  14. Yang, W. et al. Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy. Anal. Chem. 86, 6959–6967 (2014).

  15. Hang, H. C. & Bertozzi, C. R. The chemistry and biology of mucin-type O-linked glycosylation. Bioorg. Med. Chem. 13, 5021–5034 (2005).

    CAS  PubMed  Google Scholar 

  16. Yang, W., Ao, M., Hu, Y., Li, Q. K. & Zhang, H. Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO). Mol. Syst. Biol. 14, e8486 (2018).

    PubMed  PubMed Central  Google Scholar 

  17. Steentoft, C. et al. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nat. Methods 8, 977–982 (2011).

    CAS  PubMed  Google Scholar 

  18. Woo, C. M., Iavarone, A. T., Spiciarich, D. R., Palaniappan, K. K. & Bertozzi, C. R. Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat. Methods 12, 561–567 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Nilsson, J. et al. Enrichment of glycopeptides for glycan structure and attachment site identification. Nat. Methods 6, 809–811 (2009).

    CAS  PubMed  Google Scholar 

  20. Medzihradszky, K. F., Kaasik, K. & Chalkley, R. J. Tissue-specific glycosylation at the glycopeptide level. Mol. Cell Proteomics 14, 2103–2110 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Hoffmann, M., Marx, K., Reichl, U., Wuhrer, M. & Rapp, E. Site-specific O-glycosylation analysis of human blood plasma proteins. Mol. Cell Proteomics 15, 624–641 (2016).

    CAS  PubMed  Google Scholar 

  22. Mechref, Y. Use of CID/ETD mass spectrometry to analyze glycopeptides. Curr. Protoc. Protein Sci. Chapter 12, Unit 12 11 11–11 (2012).

  23. Steentoft, C. et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J. 32, 1478–1488 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Tabak, L. A. The role of mucin-type O-glycans in eukaryotic development. Semin. Cell Dev. Biol. 21, 616–621 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nat. Rev. Cancer 9, 874–885 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Sun, S. et al. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nat. Biotechnol. 34, 84 (2016).

    CAS  PubMed  Google Scholar 

  27. Zhang, H., Li, X. J., Martin, D. B. & Aebersold, R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666 (2003).

    CAS  PubMed  Google Scholar 

  28. Yang, S., Hu, Y. W., Sokoll, L. & Zhang, H. Simultaneous quantification of N- and O-glycans using a solid-phase method. Nat. Protoc. 12, 1229–1244 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Huang, J. et al. Highly efficient release of glycopeptides from hydrazide beads by hydroxylamine assisted PNGase F deglycosylation for N-glycoproteome analysis. Anal. Chem. 87, 10199–10204 (2015).

    CAS  PubMed  Google Scholar 

  30. Yang, S. et al. Deciphering protein O-glycosylation: solid-phase chemoenzymatic cleavage and enrichment. Anal. Chem. 90, 8261–8269 (2018).

    CAS  PubMed  Google Scholar 

  31. Yang, W. et al. Comparison of enrichment methods for intact N-and O-linked glycopeptides using strong anion exchange and hydrophilic interaction liquid chromatography. Anal. Chem. 89, 11193–11197 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhu, Z., Su, X., Clark, D. F., Go, E. P. & Desaire, H. Characterizing O-linked glycopeptides by electron transfer dissociation: fragmentation rules and applications in data analysis. Anal. Chem. 85, 8403–8411 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Good, D. M., Wirtala, M., McAlister, G. C. & Coon, J. J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell Proteomics 6, 1942–1951 (2007).

    CAS  PubMed  Google Scholar 

  34. Darula, Z., Sherman, J. & Medzihradszky, K. F. How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides. Mol. Cell Proteomics 11, O111 016774 (2012).

    PubMed  PubMed Central  Google Scholar 

  35. Mulagapati, S., Koppolu, V. & Raju, T. S. Decoding of O-Linked glycosylation by mass spectrometry. Biochemistry 56, 1218–1226 (2017).

    CAS  PubMed  Google Scholar 

  36. Jensen, P. H., Kolarich, D. & Packer, N. H. Mucin-type O-glycosylation-putting the pieces together. FEBS J. 277, 81–94 (2010).

    CAS  PubMed  Google Scholar 

  37. Malaker, S. A. et al. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Proc. Natl Acad. Sci. USA 116, 7278–7287 (2019).

    CAS  PubMed  Google Scholar 

  38. Khoo, K. H. Advances toward mapping the full extent of protein site-specific O-GalNAc glycosylation that better reflects underlying glycomic complexity. Curr. Opin. Struct. Biol. 56, 146–154 (2019).

    CAS  PubMed  Google Scholar 

  39. Moran, S. & Cattran, D. C. Recent advances in risk prediction, therapeutics and pathogenesis of IgA nephropathy. Minerva Med. 110, 439–449 (2019).

    PubMed  Google Scholar 

  40. Zhu, F., Li, D. & Chen, K. Structures and functions of invertebrate glycosylation. Open Biol. 9, 180232 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Haltom, A. R. & Jafar-Nejad, H. O-Linked glycans in drosophila development: overview in Glycoscience: Biology and Medicine (eds Taniguchi, N. et al.) 809–815 (Springer Japan, 2015).

  42. Sutton, C. W., O’Neill, J. A. & Cottrell, J. S. Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. Anal. Biochem. 218, 34–46 (1994).

    CAS  PubMed  Google Scholar 

  43. Muller, F., Fischer, L., Chen, Z. A., Auchynnikava, T. & Rappsilber, J. On the reproducibility of label-free quantitative cross-linking/mass spectrometry. J. Am. Soc. Mass Spectrom. 29, 405–412 (2018).

    PubMed  Google Scholar 

  44. Tabb, D. L. et al. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J. Proteome Res. 9, 761–776 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhou, J. Y. et al. Quality assessments of long-term quantitative proteomic analysis of breast cancer xenograft tissues. J. Proteome Res. 16, 4523–4530 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wu, Z. L. et al. Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology 28, 69–79 (2018).

    CAS  PubMed  Google Scholar 

  47. Dennis, G. Jr et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, P3 (2003).

    PubMed  Google Scholar 

  48. Vizcaino, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health, the National Cancer Institute, the Early Detection Research Network (EDRN, U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC, U24CA210985) and amfAR, the Foundation for AIDS Research on Bringing Bioengineers to Cure HIV (Grant amfAR 109551‐61‐RGRL).

Author information

Authors and Affiliations

  1. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Weiming Yang, Angellina Song, Minghui Ao, Yuanwei Xu & Hui Zhang

Authors
  1. Weiming Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angellina Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Minghui Ao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanwei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.Y. and H.Z. conceived the research. W.Y., A.S. and Y.X. conducted the experiments and validated the protocol. W.Y., Y.X. and M.A. conducted data analysis.

Corresponding author

Correspondence to Weiming Yang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

Yang, W. et al. Mol. Syst. Biol. 14, e8486 (2018): https://www.embopress.org/doi/full/10.15252/msb.20188486.

Supplementary information

Reporting Summary

Supplementary Data 1

Reproducibility of the EXoO protocol. O-GalNAc glycopeptides were isolated four times from the same 50 µl of human serum. O-GalNAc glycopeptides from one of the isolations were subjected to the same LC–MS/MS analysis for four times to determine the reproducibility of LC–MS/MS analysis. As a result, a total of eight LC–MS/MS runs were conducted, including four isolation repeats and four LC–MS/MS repeats of one of the isolations.

Supplementary Data 2

Quantitative performance of the EXoO protocol.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, W., Song, A., Ao, M. et al. Large-scale site-specific mapping of the O-GalNAc glycoproteome. Nat Protoc 15, 2589–2610 (2020). https://doi.org/10.1038/s41596-020-0345-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-0345-1

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research