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Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry

Nature Protocols volume 5, pages 1974–1982 (2010)Cite this article

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Abstract

The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO2) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO2 toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO2 resin. By combining TiO2 enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO2/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.

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Figure 1: Flowchart illustrating the protocol.
Figure 2
Figure 3: HILIC separation and identified sialylated glycopeptides in HeLa cell membranes.
Figure 4: Efficiency and specificity of the enrichment technique.

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References

  1. Blix, G., Lindberg, E., Odin, L. & Werner, I. Sialic acids. Nature 175, 340–341 (1955).

    Article  CAS  Google Scholar 

  2. Muhlenhoff, M., Oltmann-Norden, I., Weinhold, B., Hildebrandt, H. & Gerardy-Schahn, R. Brain development needs sugar: the role of polysialic acid in controlling NCAM functions. Biol. Chem. 390, 567–574 (2009).

    Article  Google Scholar 

  3. Cunningham, B.A. et al. Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 236, 799–806 (1987).

    Article  CAS  Google Scholar 

  4. Gorelik, E., Galili, U. & Raz, A. On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis. Cancer Metastasis Rev. 20, 245–277 (2001).

    Article  CAS  Google Scholar 

  5. Scanlin, T.F. & Glick, M.C. Terminal glycosylation and disease: influence on cancer and cystic fibrosis. Glycoconj. J. 17, 617–626 (2000).

    Article  CAS  Google Scholar 

  6. Fukuda, M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res. 56, 2237–2244 (1996).

    CAS  PubMed  Google Scholar 

  7. Kim, Y.J. & Varki, A. Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj. J. 14, 569–576 (1997).

    Article  CAS  Google Scholar 

  8. Yogeeswaran, G. & Salk, P.L. Metastatic potential is positively correlated with cell surface sialylation of cultured murine tumor cell lines. Science 212, 1514–1516 (1981).

    Article  CAS  Google Scholar 

  9. Rempel, H., Calosing, C., Sun, B. & Pulliam, L. Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity. PLoS One 3, e1967 (2008).

    Article  Google Scholar 

  10. Fozzard, H.A. & Kyle, J.W. Do defects in ion channel glycosylation set the stage for lethal cardiac arrhythmias? Sci. STKE 2002, pe19 (2002).

    PubMed  Google Scholar 

  11. Ufret-Vincenty, C.A., Baro, D.J. & Santana, L.F. Differential contribution of sialic acid to the function of repolarizing K(+) currents in ventricular myocytes. Am. J. Physiol. Cell Physiol. 281, C464–474 (2001).

    Article  CAS  Google Scholar 

  12. Schauer, R. Chemistry, metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem. 40, 131–234 (1982).

    Article  CAS  Google Scholar 

  13. Tanner, E.M. The enzymes of sialic acid biosynthesis. Bioorg. Chem. 33, 216–228 (2005).

    Article  CAS  Google Scholar 

  14. Munster-Kuhnel, A.K. et al. Structure and function of vertebrate CMP-sialic acid synthetases. Glycobiology 14, 43R–51R (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Zeng, Y., Ramya, T.N., Dirksen, A., Dawson, P.E. & Paulson, J.C. High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6, 207–209 (2009).

    Article  CAS  Google Scholar 

  17. Mahal, L.K., Yarema, K.J. & Bertozzi, C.R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).

    Article  CAS  Google Scholar 

  18. Saxon, E. et al. Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J. Am. Chem. Soc. 124, 14893–14902 (2002).

    Article  CAS  Google Scholar 

  19. Prescher, J.A., Dube, D.H. & Bertozzi, C.R. Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877 (2004).

    Article  CAS  Google Scholar 

  20. Bond, M.R. & Kohler, J.J. Chemical methods for glycoprotein discovery. Curr. Opin. Chem. Biol. 11, 52–58 (2007).

    Article  CAS  Google Scholar 

  21. Laughlin, S.T., Baskin, J.M., Amacher, S.L. & Bertozzi, C.R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    Article  CAS  Google Scholar 

  22. Cummings, R.D. & Kornfeld, S. Fractionation of asparagine-linked oligosaccharides by serial lectin-Agarose affinity chromatography. A rapid, sensitive, and specific technique. J. Biol. Chem. 257, 11235–11240 (1982).

    CAS  PubMed  Google Scholar 

  23. Yang, Z. & Hancock, W.S. Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography. J. Chromatogr. A 1070, 57–64 (2005).

    Article  CAS  Google Scholar 

  24. Zielinska, D.F., Gnad, F., Wisniewski, J.R. & Mann, M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141, 897–907 (2010).

    Article  CAS  Google Scholar 

  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. Yodoshi, M., Ikuta, T., Mouri, Y. & Suzuki, S. Specific extraction of sialic-acid-containing glycans and glycopeptides using serotonin-bonded silica. Anal. Sci. 26, 75–81 (2010).

    Article  CAS  Google Scholar 

  27. Sturgeon, R.J. & Sturgeon, C.M. Affinity chromatography of sialoglycoproteins, utilising the interaction of serotonin with n-acetylneuraminic acid and its derivatives. Carbohydr. Res. 103, 213–219 (1982).

    Article  CAS  Google Scholar 

  28. Lewandrowski, U., Zahedi, R.P., Moebius, J., Walter, U. & Sickmann, A. Enhanced N-glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranes. Mol. Cell Proteomics 6, 1933–1941 (2007).

    Article  CAS  Google Scholar 

  29. Wuhrer, M., Koeleman, C.A., Hokke, C.H. & Deelder, A.M. Protein glycosylation analyzed by normal-phase nano-liquid chromatography—mass spectrometry of glycopeptides. Anal. Chem. 77, 886–894 (2005).

    Article  CAS  Google Scholar 

  30. Thaysen-Andersen, M., Engholm-Keller, K. & Roepstorff, P. (eds.) Analysis of Protein Glycosylation and Phosphorylation using HILIC-MS (Taylor & Francis, 2010).

  31. Takegawa, Y. et al. Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography. J. Sep. Sci. 29, 2533–2540 (2006).

    Article  CAS  Google Scholar 

  32. Boersema, P.J., Divecha, N., Heck, A.J. & Mohammed, S. Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy. J. Proteome Res. 6, 937–946 (2007).

    Article  CAS  Google Scholar 

  33. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics 4, 873–886 (2005).

    Article  CAS  Google Scholar 

  34. Larsen, M.R., Jensen, S.S., Jakobsen, L.A. & Heegaard, N.H. Exploring the sialiome using titanium dioxide chromatography and mass spectrometry. Mol. Cell Proteomics 6, 1778–1787 (2007).

    Article  CAS  Google Scholar 

  35. Roddick-Lanzilotta, A.D. & McQuillan, A.J. An in situ infrared spectroscopic study of glutamic acid and of aspartic acid adsorbed on TiO(2): implications for the biocompatibility of titanium. J. Colloid Interface Sci. 227, 48–54 (2000).

    Article  CAS  Google Scholar 

  36. Fujiki, Y., Hubbard, A.L., Fowler, S. & Lazarow, P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102 (1982).

    Article  CAS  Google Scholar 

  37. Tretter, V., Altmann, F. & Marz, L. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1–3 to the asparagine-linked N-acetylglucosamine residue. Eur. J. Biochem. 199, 647–652 (1991).

    Article  CAS  Google Scholar 

  38. Mueller, L.N., Brusniak, M.Y., Mani, D.R. & Aebersold, R. An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J. Proteome Res. 7, 51–61 (2008).

    Article  CAS  Google Scholar 

  39. O'Hanlon, D.M. et al. Soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in breast carcinoma. Eur. J. Cancer 38, 2252–2257 (2002).

    Article  CAS  Google Scholar 

  40. Segu, Z.M. & Mechref, Y. Characterizing protein glycosylation sites through higher-energy C-trap dissociation. Rapid. Commun. Mass Spectrom. 24, 1217–1225 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by the Lundbeck Foundation (to M.R.L.; Junior Group Leader Fellowship), the Danish Natural Science Research Council (to M.R.L.; 09-06-5989) and the National Health and Medical Research Council (NHMRC) of Australia (to S.J.C.; 571002).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Southern Denmark, Odense, Denmark

    Giuseppe Palmisano, Sara Eun Lendal, Kasper Engholm-Keller, Benjamin L Parker & Martin R Larsen

  2. Department of Molecular Medicine, Cancer and Inflammation Research, Odense University Hospital, Odense, Denmark

    Rikke Leth-Larsen

  3. Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney, New South Wales, Australia

    Benjamin L Parker

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  1. Giuseppe Palmisano
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Contributions

G.P. optimized the protocol, performed the experiments and wrote the manuscript; S.E.L. performed the experiments; K.E.-K. set up the HILIC fractionation; R.L.-L. proofread the manuscript; B.L.P. wrote the manuscript; M.R.L. conceived the idea, supervised the experiments and wrote the manuscript.

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Correspondence to Martin R Larsen.

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The authors declare no competing financial interests.

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Palmisano, G., Lendal, S., Engholm-Keller, K. et al. Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry. Nat Protoc 5, 1974–1982 (2010). https://doi.org/10.1038/nprot.2010.167

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