Advertisement

Titanium Dioxide Enrichment of Sialic Acid-Containing Glycopeptides

  • Giuseppe Palmisano
  • Sara E. Lendal
  • Martin R. LarsenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 753)

Abstract

Glycosylation is one of the many post-translational protein modifications that regulate several biological processes of proteins and lipids. In particular aberrant sialylation, at the terminal position of the glycan structures of cell surface proteins, occurs in numerous diseases such as cancer metastasis and viral infections. Methodological improvements in the sample preparation and analysis currently enable the detailed identification of the glycosylation sites and glycan structure characterization. In this context, the aim of this chapter is to describe a methodology to identify the glycosylation site of N-linked sialylated glycoproteins. The method relies on the specificity of titanium dioxide affinity chromatography to isolate sialic acid-containing glycopeptides. After enzymatic release of the glycans, the enriched sialylated glycopeptides are analyzed by mass spectrometry. This strategy was applied to a crude membrane fraction of EGF-stimulated HeLa cells metabolically labeled with SILAC enabling both qualitative and quantitative analyses of sialoglycopeptides.

Key words

Sialylation glycosylation titanium dioxide quantitative proteomics mass spectrometry 

References

  1. 1.
    Lowe, J. B., and Marth, J. D. (2003) A genetic approach to mammalian glycan function, Annu Rev Biochem 72, 643–691.PubMedCrossRefGoogle Scholar
  2. 2.
    Apweiler, R., Hermjakob, H., and Sharon, N. (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database, Biochim Biophys Acta 1473, 4–8.PubMedGoogle Scholar
  3. 3.
    Ohtsubo, K., and Marth, J. D. (2006) Glycosylation in cellular mechanisms of health and disease, Cell 126, 855–867.PubMedCrossRefGoogle Scholar
  4. 4.
    Varki, A., Cummings, R., Esko, J. D., Freeze, H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E. (2008) Essentials in Glycobiology, Cold Spring Harbor Press, Cold Spring Harbor, NY.Google Scholar
  5. 5.
    Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct, Glycobiology 3, 97–130.PubMedCrossRefGoogle Scholar
  6. 6.
    Hart, G. W., Housley, M. P., and Slawson, C. (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins, Nature 446, 1017–1022.PubMedCrossRefGoogle Scholar
  7. 7.
    Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system, Science 291, 2370–2376.PubMedCrossRefGoogle Scholar
  8. 8.
    Freeze, H. H. (2006) Genetic defects in the human glycome, Nat Rev Genet 7, 537–551.PubMedCrossRefGoogle Scholar
  9. 9.
    Freeze, H. H., and Aebi, M. (2005) Altered glycan structures: the molecular basis of congenital disorders of glycosylation, Curr Opin Struct Biol 15, 490–498.PubMedCrossRefGoogle Scholar
  10. 10.
    Kim, Y. J., and Varki, A. (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer, Glycoconj J 14, 569–576.PubMedCrossRefGoogle Scholar
  11. 11.
    Kornfeld, R., and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides, Annu Rev Biochem 54, 631–664.PubMedCrossRefGoogle Scholar
  12. 12.
    Hart, G. W. (1992) Glycosylation, Curr Opin Cell Biol 4, 1017–1023.PubMedCrossRefGoogle Scholar
  13. 13.
    Low, M. G. (1989) Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins, FASEB J 3, 1600–1608.PubMedGoogle Scholar
  14. 14.
    Haynes, P. A. (1998) Phosphoglycosylation: a new structural class of glycosylation? Glycobiology 8, 1–5.PubMedCrossRefGoogle Scholar
  15. 15.
    Hartmann, S., and Hofsteenge, J. (2000) Properdin, the positive regulator of complement, is highly C-mannosylated, J Biol Chem 275, 28569–28574.PubMedCrossRefGoogle Scholar
  16. 16.
    Schauer, R. (2000) Achievements and challenges of sialic acid research, Glycoconj J 17, 485–499.PubMedCrossRefGoogle Scholar
  17. 17.
    Varki, N. M., and Varki, A. (2007) Diversity in cell surface sialic acid presentations: implications for biology and disease, Lab Invest 87, 851–857.PubMedCrossRefGoogle Scholar
  18. 18.
    Thomas, P. (1996) Cell surface sialic acid as a mediator of metastatic potential in colorectal cancer, Cancer J 9, 1–10.Google Scholar
  19. 19.
    Fozzard, H. A., and Kyle, J. W. (2002) Do defects in ion channel glycosylation set the stage for lethal cardiac arrhythmias? Sci STKE 2002, pe19.CrossRefGoogle Scholar
  20. 20.
    Rhim, A. D., Stoykova, L. I., Trindade, A. J., Glick, M. C., and Scanlin, T. F. (2004) Altered terminal glycosylation and the pathophysiology of CF lung disease, J Cyst Fibros 3 (Suppl 2), 95–96.PubMedCrossRefGoogle Scholar
  21. 21.
    Goodarzi, M. T. (2008) Changes in sialylation of low-density lipoprotein in coronary artery disease, Eur J Lipid Sci Tech 110, 302–306.CrossRefGoogle Scholar
  22. 22.
    Coppo, R., and Amore, A. (2004) Aberrant glycosylation in IgA nephropathy (IgAN), Kidney Int 65, 1544–1547.PubMedCrossRefGoogle Scholar
  23. 23.
    Stray, S. J., Cummings, R. D., and Air, G. M. (2000) Influenza virus infection of desialylated cells, Glycobiology 10, 649–658.PubMedCrossRefGoogle Scholar
  24. 24.
    Zhao, J., Simeone, D. M., Heidt, D., Anderson, M. A., and Lubman, D. M. (2006) Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum, J Proteome Res 5, 1792–1802.PubMedCrossRefGoogle Scholar
  25. 25.
    Yang, Z., and Hancock, W. S. (2005) Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography, J Chromatogr A 1070, 57–64.PubMedCrossRefGoogle Scholar
  26. 26.
    Zeng, Y., Ramya, T. N., Dirksen, A., Dawson, P. E., and Paulson, J. C. (2009) High-efficiency labeling of sialylated glycoproteins on living cells, Nat Methods 6, 207–209.PubMedCrossRefGoogle Scholar
  27. 27.
    Nilsson, J., Ruetschi, U., Halim, A., Hesse, C., Carlsohn, E., Brinkmalm, G., and Larson, G. (2009) Enrichment of glycopeptides for glycan structure and attachment site identification, Nat Methods 6, 809–811.PubMedCrossRefGoogle Scholar
  28. 28.
    Lewandrowski, U., Zahedi, R. P., Moebius, J., Walter, U., and Sickmann, A. (2007) Enhanced N-glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes, Mol Cell Proteomics 6, 1933–1941.PubMedCrossRefGoogle Scholar
  29. 29.
    Larsen, M. R., Jensen, S. S., Jakobsen, L. A., and Heegaard, N. H. (2007) Exploring the sialiome using titanium dioxide chromatography and mass spectrometry, Mol Cell Proteomics 6, 1778–1787.PubMedCrossRefGoogle Scholar
  30. 30.
    Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum, J Cell Biol 93, 97–102.PubMedCrossRefGoogle Scholar
  31. 31.
    Mueller, L. N., Brusniak, M. Y., Mani, D. R., and Aebersold, R. (2008) An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data, J Proteome Res 7, 51–61.PubMedCrossRefGoogle Scholar
  32. 32.
    Cox, J., and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification, Nat Biotechnol 26, 1367–1372.PubMedCrossRefGoogle Scholar
  33. 33.
    Ong, S. E., and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC), Nat Protoc 1, 2650–2660.PubMedCrossRefGoogle Scholar
  34. 34.
    Pizzio, L. R. (2005) Mesoporous titania: effect of thermal treatment on the texture and acidic properties, Mater Lett. 59, 994.CrossRefGoogle Scholar
  35. 35.
    Angel, P. M., Lim, J. M., Wells, L., Bergmann, C., and Orlando, R. (2007) A potential pitfall in 18O-based N-linked glycosylation site mapping, Rapid Commun Mass Spectrom 21, 674–682.PubMedCrossRefGoogle Scholar
  36. 36.
    Larsen, M. R. (2003) Mass spectrometric characterization of posttranslationally modified proteins-phosphorylation, Methods Mol Biol 251, 245.Google Scholar
  37. 37.
    Larsen, M. R., Cordwell, S. J., Roepstorff, P. (2002) Graphite powder as an alternative to reversed phase material for desalting and concentration of peptide mixtures prior to mass spectrometric analysis, Proteomics 2, 1277–1287.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Giuseppe Palmisano
    • 1
  • Sara E. Lendal
    • 1
  • Martin R. Larsen
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

Personalised recommendations