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.
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References
Lowe, J. B., and Marth, J. D. (2003) A genetic approach to mammalian glycan function, Annu Rev Biochem 72, 643–691.
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.
Ohtsubo, K., and Marth, J. D. (2006) Glycosylation in cellular mechanisms of health and disease, Cell 126, 855–867.
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.
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct, Glycobiology 3, 97–130.
Hart, G. W., Housley, M. P., and Slawson, C. (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins, Nature 446, 1017–1022.
Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system, Science 291, 2370–2376.
Freeze, H. H. (2006) Genetic defects in the human glycome, Nat Rev Genet 7, 537–551.
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.
Kim, Y. J., and Varki, A. (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer, Glycoconj J 14, 569–576.
Kornfeld, R., and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides, Annu Rev Biochem 54, 631–664.
Hart, G. W. (1992) Glycosylation, Curr Opin Cell Biol 4, 1017–1023.
Low, M. G. (1989) Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins, FASEB J 3, 1600–1608.
Haynes, P. A. (1998) Phosphoglycosylation: a new structural class of glycosylation? Glycobiology 8, 1–5.
Hartmann, S., and Hofsteenge, J. (2000) Properdin, the positive regulator of complement, is highly C-mannosylated, J Biol Chem 275, 28569–28574.
Schauer, R. (2000) Achievements and challenges of sialic acid research, Glycoconj J 17, 485–499.
Varki, N. M., and Varki, A. (2007) Diversity in cell surface sialic acid presentations: implications for biology and disease, Lab Invest 87, 851–857.
Thomas, P. (1996) Cell surface sialic acid as a mediator of metastatic potential in colorectal cancer, Cancer J 9, 1–10.
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.
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.
Goodarzi, M. T. (2008) Changes in sialylation of low-density lipoprotein in coronary artery disease, Eur J Lipid Sci Tech 110, 302–306.
Coppo, R., and Amore, A. (2004) Aberrant glycosylation in IgA nephropathy (IgAN), Kidney Int 65, 1544–1547.
Stray, S. J., Cummings, R. D., and Air, G. M. (2000) Influenza virus infection of desialylated cells, Glycobiology 10, 649–658.
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.
Yang, Z., and Hancock, W. S. (2005) Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography, J Chromatogr A 1070, 57–64.
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.
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.
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.
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.
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.
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.
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.
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.
Pizzio, L. R. (2005) Mesoporous titania: effect of thermal treatment on the texture and acidic properties, Mater Lett. 59, 994.
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.
Larsen, M. R. (2003) Mass spectrometric characterization of posttranslationally modified proteins-phosphorylation, Methods Mol Biol 251, 245.
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.
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Palmisano, G., Lendal, S.E., Larsen, M.R. (2011). Titanium Dioxide Enrichment of Sialic Acid-Containing Glycopeptides. In: Gevaert, K., Vandekerckhove, J. (eds) Gel-Free Proteomics. Methods in Molecular Biology, vol 753. Humana Press. https://doi.org/10.1007/978-1-61779-148-2_21
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DOI: https://doi.org/10.1007/978-1-61779-148-2_21
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