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Structural analysis of N- and O-glycans released from glycoproteins

Nature Protocols volume 7, pages 1299–1310 (2012)Cite this article

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Abstract

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

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Figure 1: Screening of N-linked glycans from erythropoietin.
Figure 2: Screening of reduced N-linked and O-linked glycans from erythropoietin.
Figure 3: PGC LC-MS N-glycan profile derived from 5 μg of human IgG.
Figure 4: Separation by LC enables individual tandem-MS spectra acquisition for individual glycan structures.
Figure 5

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References

  1. Dennis, J.W., Granovsky, M. & Warren, C.E. Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta 1473, 21–34 (1999).

    Article  CAS  Google Scholar 

  2. Otake, Y. et al. Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer. J. Biochem. 129, 537–542 (2001).

    Article  CAS  Google Scholar 

  3. Karlsson, N.G. et al. Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis. Biochem. J. 350, 805–814 (2000).

    Article  CAS  Google Scholar 

  4. Landberg, E. et al. Changes in glycosylation of human bile-salt-stimulated lipase during lactation. Arch. Biochem. Biophys. 377, 246–254 (2000).

    Article  CAS  Google Scholar 

  5. Nakano, M., Saldanha, R., Gobel, A., Kavallaris, M. & Packer, N.H. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells. Mol. Cell Proteomics 10, M111 009001 (2011).

    Article  Google Scholar 

  6. Wilson, N.L. et al. Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes. J. Proteome Res. 7, 3687–3696 (2008).

    Article  CAS  Google Scholar 

  7. Aggarwal, S. What's fueling the biotech engine? Nat. Biotechnol. 25, 1097–1104 (2007).

    Article  CAS  Google Scholar 

  8. Maiorella, B.L. et al. Effect of culture conditions on IgM antibody structure, pharmacokinetics and activity. Biotechnology (N Y) 11, 387–392 (1993).

    Article  CAS  Google Scholar 

  9. Moll, M., Kaufmann, A. & Maisner, A. Influence of N-glycans on processing and biological activity of the nipah virus fusion protein. J. Virol. 78, 7274–7278 (2004).

    Article  CAS  Google Scholar 

  10. Morais, V.A., Costa, M.T. & Costa, J. N-Glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells. Biochim. Biophys. Acta 1619, 133–138 (2003).

    Article  CAS  Google Scholar 

  11. Kolarich, D., Jensen, P.H., Altmann, F. & Packer, N.H. Determination of site-specific glycan heterogeneity on glycoproteins. Nat. Protoc. 7, 1285–1298 (2012).

    Article  CAS  Google Scholar 

  12. Wilson, N.L., Schulz, B.L., Karlsson, N.G. & Packer, N.H. Sequential analysis of N- and O-linked glycosylation of 2D-PAGE–separated glycoproteins. J. Proteome Res. 1, 521–529 (2002).

    Article  CAS  Google Scholar 

  13. Schulz, B.L., Packer, N.H. & Karlsson, N.G. Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. Anal. Chem. 74, 6088–6097 (2002).

    Article  CAS  Google Scholar 

  14. Karlsson, N.G. et al. Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Commun. Mass Spectrom. 18, 2282–2292 (2004).

    Article  CAS  Google Scholar 

  15. Karlsson, N.G., Schulz, B.L. & Packer, N.H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 15, 659–672 (2004).

    Article  CAS  Google Scholar 

  16. Schulz, B.L. et al. Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions. Biochem. J. 387, 911–919 (2005).

    Article  CAS  Google Scholar 

  17. Schulz, B.L. et al. Glycosylation of sputum mucins is altered in cystic fibrosis patients. Glycobiology 17, 698–712 (2007).

    Article  CAS  Google Scholar 

  18. Estrella, R.P., Whitelock, J.M., Packer, N.H. & Karlsson, N.G. Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan. Anal. Chem. 79, 3597–3606 (2007).

    Article  CAS  Google Scholar 

  19. Thomson, K.A., Schulz, B.L., Packer, N.H. & Karlsson, N.G. MUC5B glycosylation in human saliva reflects blood group and secretor status. Glycobiology 15, 791–804 (2005).

    Article  Google Scholar 

  20. Wada, Y. et al. Comparison of the methods for profiling glycoprotein glycans—HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study. Glycobiology 17, 411–422 (2007).

    Article  CAS  Google Scholar 

  21. Wada, Y. et al. Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1. Mol. Cell Proteomics 9, 719–727 (2010).

    Article  CAS  Google Scholar 

  22. Wilson, N.L. et al. Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes. J. Proteome Res. 7, 3687–3696 (2008).

    Article  CAS  Google Scholar 

  23. Deshpande, N., Jensen, P.H., Packer, N.H. & Kolarich, D. GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA. J. Proteome Res. 9, 1063–1075 (2010).

    Article  CAS  Google Scholar 

  24. Lee, A. et al. Liver membrane proteome glycosylation changes in mice bearing an extra-hepatic tumour. Mol. Cell Proteomics doi:10.1074/mcp.M900538-MCP200 (2010).

  25. Lee, A. et al. The lectin riddle: glycoproteins fractionated from complex mixtures have similar glycomic profiles. OMICS 14, 487–499 (2010).

    Article  Google Scholar 

  26. Natsuka, S. & Hase, S. Analysis of N- and O-glycans by pyridylamination. Methods Mol. Biol. 76, 101–113 (1998).

    CAS  PubMed  Google Scholar 

  27. Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B. & Dwek, R.A. A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240, 210–226 (1996).

    Article  CAS  Google Scholar 

  28. Rudd, P.M. et al. Oligosaccharide sequencing technology. Nature 388, 205–207 (1997).

    Article  CAS  Google Scholar 

  29. Ceroni, A. et al. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659 (2008).

    Article  CAS  Google Scholar 

  30. Goldberg, D. et al. Automated N-glycopeptide identification using a combination of single- and tandem-MS. J. Proteome Res. 6, 3995–4005 (2007).

    Article  CAS  Google Scholar 

  31. Jang-Lee, J. Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies. et al. in Methods in Enzymology Vol. 415 (ed. Fukuda, M.) 59–86 (Academic Press, 2006).

  32. Dell, A. & Morris, H.R. Glycoprotein structure determination by mass spectrometry. Science 291, 2351–2356 (2001).

    Article  CAS  Google Scholar 

  33. Stumpo, K.A. & Reinhold, V.N. The N-glycome of human plasma. J. Proteome Res. 9, 4823–4830 (2010).

    Article  CAS  Google Scholar 

  34. Lei, M., Mechref, Y. & Novotny, M.V. Structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide. J. Am. Soc. Mass Spectrom. 20, 1660–1671 (2009).

    Article  CAS  Google Scholar 

  35. Srikrishna, G. et al. A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses. J. Immunol. 166, 624–632 (2001).

    Article  CAS  Google Scholar 

  36. Wuhrer, M., de Boer, A.R. & Deelder, A.M. Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry. Mass Spectrom. Rev. 28, 192–206 (2009).

    Article  CAS  Google Scholar 

  37. Lee, A. et al. Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture. J. Proteome Res. 8, 770–781 (2009).

    Article  CAS  Google Scholar 

  38. Schulz, B.L. et al. Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions. Biochem. J. 387, 911–919 (2005).

    Article  CAS  Google Scholar 

  39. Harvey, D.J. Matrix-assisted laser desorption/ionization mass spectrometry of sphingo- and glycosphingo-lipids. J. Mass Spectrom. 30, 1311–1324 (1995).

    Article  CAS  Google Scholar 

  40. Zaia, J. Mass spectrometry and glycomics. OMICS 14, 401–418 (2010).

    Article  CAS  Google Scholar 

  41. Kawasaki, N., Ohta, M., Hyuga, S., Hyuga, M. & Hayakawa, T. Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin. Anal. Biochem. 285, 82–91 (2000).

    Article  CAS  Google Scholar 

  42. Pabst, M., Bondili, J.S., Stadlmann, J., Mach, L. & Altmann, F. Mass plus retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans. Anal. Chem. 79, 5051–5057 (2007).

    Article  CAS  Google Scholar 

  43. Wada, Y. et al. Comparison of methods for profiling O-glycosylation. Mol. Cell. Proteomics 9, 719–727 (2010).

    Article  CAS  Google Scholar 

  44. Wuhrer, M., Koeleman, C.A., Hokke, C.H. & Deelder, A.M. Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments. Rapid Commun. Mass Spectrom.: RCM 20, 1747–1754 (2006).

    Article  CAS  Google Scholar 

  45. Hayes, C.A. et al. UniCarb-DB: a database resource for glycomic discovery. Bioinformatics 27, 1343–1344 (2011).

    Article  CAS  Google Scholar 

  46. Campbell, M.P. et al. UniCarbKB: putting the pieces together for glycomics research. Proteomics 11, 4117–4121 (2011).

    Article  CAS  Google Scholar 

  47. Ito, H., Kameyama, A., Sato, T. & Narimatsu, H. Preparation of a glycan library using a variety of glycosyltrasferases. Methods Mol. Biol. 534, 283–291 (2009).

    CAS  PubMed  Google Scholar 

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Acknowledgements

P.H.J. was supported by the Danish Agency for Science, Technology and Innovation (grant 272-07-0066). D.K. was supported by an Erwin Schrödinger Fellowship from the Austrian Science Fund (grant J2661) and Macquarie University. We also thank M. Nakano for the preparation of Figure 5 (based on data from ref. 5).

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Author notes

  1. Pia H Jensen & Daniel Kolarich

    Present address: Present address: Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark (P.H.J.); Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany (D.K.).,

Authors and Affiliations

  1. Faculty of Science, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, New South Wales, Australia

    Pia H Jensen, Daniel Kolarich & Nicolle H Packer

  2. Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden

    Niclas G Karlsson

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Contributions

All authors contributed equally to this work. N.G.K. developed and validated the initial protocol and performed the analysis of recombinant EPO. P.H.J. tested, optimized and wrote the protocol. D.K. and N.H.P. co-wrote and edited the final manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding author

Correspondence to Nicolle H Packer.

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Jensen, P., Karlsson, N., Kolarich, D. et al. Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 7, 1299–1310 (2012). https://doi.org/10.1038/nprot.2012.063

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