Abstract
Glycoproteins constitute a large fraction of the proteome. The fundamental role of protein glycosylation in cellular development, growth, and differentiation, tissue development, and in host-pathogen interactions is by now widely accepted. Proteome-wide characterization of glycoproteins is a complex task and is currently achieved by mass spectrometry-based methods that enable identification of glycoproteins and localization, classification, and analysis of individual glycan structures on proteins. In this chapter we briefly introduce a range of analytical technologies for recovery and analysis of glycoproteins and glycopeptides. Combinations of affinity-enrichment techniques, chemical and biochemical protocols, and advanced mass spectrometry facilitate detailed glycoprotein analysis in proteomics, from fundamental biological studies to biomarker discovery in biomedicine.
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References
Jensen, O. N. (2006) Interpreting the protein language using proteomics. Nat. Rev. Mol. Cell. Biol., 7, 391–403.
Sharon, N. and Lis, H. (1997) Glycoproteins: structure and function. In Glycosciences: Status and Perspectives (Gabius, H.-J., Gabius, S., eds.). Chapman & Hall, Wienheim, Germany, pp. 133–162.
Varki, A. (1993) Biological roles of oligosaccharides—all of the theories are correct. Glycobiology 3, 97–130.
Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G., and Marth, J. (eds.). (1999) Essentials of Glycobiology. Cold Spring Harbor Press, Cold Spring Harbor, NY.
Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364–2369.
Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system. Science 291, 2370–2376.
Wells, L., Vosseller, K. and Hart, G. W. (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376–2378.
Hofsteenge, J., Muller, D. R., Debeer, T., Loffler, A., Richter, W. J., and Vliegenthart, J. F. G. (1994) New-type of linkage between a carbohydrate and a protein— C-glycosylation of a specific tryptophan residue in human Rnase U-S. Biochemistry 33, 13524–13530.
Harvey, D. J. (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom. Rev. 18, 349–450.
Medzihradszky, K. F. (2005) Characterization of protein N-glycosylation. Methods Enzymol. 405, 116–138.
Mortz, E., Sareneva, T., Julkunen, I., and Roepstorff, P. (1996) Does matrix-assisted laser desorption/ionization mass spectrometry allow analysis of carbohydrate heterogeneity in glycoproteins? A study of natural human interferon-gamma. J. Mass Spectrom. 31, 1109–1118.
Gabius, H. J., Andre, S., Kaltner, H., and Siebert, H.C. (2002) The sugar code: functional lectinomics. Biochim. Biophys. Acta 1572, 165–177.
Wang, Y., Wu, S. L., and Hancock, W. S. (2006) Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS). Biotechnol. Prog. 22, 873–880.
Drake, R. R., Schwegler, E. E., Malik, G., Diaz, J. I., Block, T., Mehta, A., and Semmes, O. J. (2006) Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers. Mol. Cell. Proteomics 5, 1957–1967.
Peracaula, R., Royle, L., Tabares, G., Mallorqui-Fernandez, G., Barrabes, S., Harvey, D. J., Dwek, R. A., Rudd, P. M., and de Llorens, R. (2003) Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states. Glycobiology 13, 227–244.
Larsen, M. R., Cordwell, S. J., and Roepstorff, P. (2002) Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry. Proteomics 2, 1277–1287.
Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N., and Roepstorff, P. (2004) A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J. Proteome Res. 3, 556–566.
Omaetxebarria, M. J., Hagglund, P., Elortza, F., Hooper, N. M., Arizmendi, J. M., and Jensen, O. N. (2006) Isolation and characterization of glycosylphosphatidylinositol-anchored peptides by hydrophilic interaction chromatography and MALDI tandem mass spectrometry. Anal. Chem. 78, 3335–3341.
Hagglund, P., Matthiesen, R., Elortza, F., Hojrup, P., Roepstorff, P., Jensen, O. N., and Bunkenborg, J. (2007) An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins. J. Proteome Res. 6, 3021–3031.
Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. D. (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886.
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.
Bunkenborg, J., Hägglund, P., and Jensen, O. N. (2007) Modification-specific proteomic analysis of glycoproteins in human body fluids by mass spectrometry. In Proteomics of Human Body Fluids: Principles Methods, and Applications (Thongboonkerd, V., ed.), Humana Press, Totowa, NJ.
Zhang, H., Li, X. J., Martin, D. B., and Aebersold, R. (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666.
Zhang, H. and Aebersold, R. (2006) Isolation of glycoproteins and identification of their N-linked glycosylation sites. In New and Emerging Proteomic Techniques (Nedelkov, D., Nelson, R. W., eds.), Vol. 328, pp. 177–185. Humana Press, Totowa, NJ
Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M., Hirabayashi, J., Kasai, K., Takahashi, N., and Isobe, T. (2003) Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nat. Biotechnol. 21, 667–672.
Harvey, D. J. (2005) Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Proteomics 5, 1774–1786.
Rappsilber, J., Ishihama, Y., and Mann, M. (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem, 75, 663–670.
Yang, Z. P. and Hancock, W. S. (2004) Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column. J. Chromatogr. A 1053, 79–88.
Cummings, R. D. (1997) Lectins as tools for glycoconjugate purification and characterization. In Glycosciences: Status and Perspectives (Gabius, H.-J., Gabius, S., eds.), pp. 191–199. Chapman & Hall, Wienheim, Germany
Gabius, H. J., Siebert, H. C., Andre, S., Jimenez-Barbero, J., and Rudiger, H. (2004) Chemical biology of the sugar code. Chembiochemistry 5, 741–764.
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Selby, D.S., Larsen, M.R., Calvano, C.D., Jensen, O.N. (2008). Identification and Characterization of N-Glycosylated Proteins Using Proteomics. In: Thompson, J.D., Ueffing, M., Schaeffer-Reiss, C. (eds) Functional Proteomics. Methods in Molecular Biology, vol 484. Humana Press. https://doi.org/10.1007/978-1-59745-398-1_17
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DOI: https://doi.org/10.1007/978-1-59745-398-1_17
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