Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated. Glycosylation plays a role in protein folding, interaction, stability, and mobility, as well as in signal transduction. Thus, by regulating protein activity, glycosylation is involved in the normal functioning of the cell and in the development of diseases. Indeed, in the past few decades there has been a growing realization of the importance of protein glycosylation, as aberrant glycosylation has been implicated in metabolic, neurodegenerative, and neoplastic diseases. Thus, the identification and quantification of protein-borne oligosaccharides have become increasingly important both in the basic sciences of biochemistry and glycobiology and in the applicative sciences, particularly biomedicine and biotechnology. Here, we review the state-of-the-art methodologies for the identification and quantification of oligosaccharides, specifically N- and O-glycosylated proteins. 1. Protein Glycosylation Carbohydrates are essential for cell metabolism and energy production and are building blocks of the extracellular matrix. Carbohydrates also play a role in a variety of biological processes, such as intra- and intercellular signaling and mediation of cell-cell interactions. In plants and animals alike, they may be found in various forms and structures—monosaccharides, oligosaccharides, or long and complex polysaccharides. Saccharides may be present in their free forms or they may be linked to other molecules, such as proteins and lipids. In proteoglycans the saccharide is the dominant component, whereas in glycoproteins, the saccharide constitutes only a small portion of the entire molecule. Protein glycosylation, that is, the attachment of a saccharide moiety to a protein, is a modification that occurs either cotranslationally or posttranslationally. The two major types of glycosylation, N-linked and O-linked, are both involved in the maintenance of protein conformation and activity, in protein protection from proteolytic degradation, and in protein intracellular trafficking and secretion [1]. N-glycan moieties also play a key role in the folding, processing, and secretion of proteins from the endoplasmic reticulum (ER) and the Golgi apparatus [1]. In the past few decades, growing attention has been directed to protein glycosylation in the biomedical field, since aberrant glycosylations have been associated with various congenital [2], metabolic, neurodegenerative [3], and
References
[1]
A. Varki, “Biological roles of oligosaccharides: all of the theories are correct,” Glycobiology, vol. 3, no. 2, pp. 97–130, 1993.
[2]
E. A. Eklund and H. H. Freeze, “The congenital disorders of glycosylation: a multifaceted group of syndromes,” NeuroRx, vol. 3, no. 2, pp. 254–263, 2006.
[3]
T. Lefebvre, C. Guinez, V. Dehennaut, O. Beseme-Dekeyser, W. Morelle, and J. C. Michalski, “Does O-GlcNAc play a role in neurodegenerative diseases?” Expert Review of Proteomics, vol. 2, no. 2, pp. 265–275, 2005.
[4]
P. M. Rudd, T. Elliott, P. Cresswell, I. A. Wilson, and R. A. Dwek, “Glycosylation and the immune system,” Science, vol. 291, no. 5512, pp. 2370–2376, 2001.
[5]
M. E. Taylor and K. Drickamer, Introduction to Glycobiology, Oxford University Press, New York, NY, USA, 2nd edition, 2006.
[6]
S. Dube, J. W. Fisher, and J. S. Powell, “Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function,” Journal of Biological Chemistry, vol. 263, no. 33, pp. 17516–17521, 1988.
[7]
A. Helenius and M. Aebi, “Intracellular functions of N-linked glycans,” Science, vol. 291, no. 5512, pp. 2364–2369, 2001.
[8]
L. Ellgaard, M. Molinari, and A. Helenius, “Setting the standards: quality control in the secretory pathway,” Science, vol. 286, no. 5446, pp. 1882–1888, 1999.
[9]
A. Varki, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, New York, NY, USA, 2nd edition, 2009.
[10]
L. A. Tabak, “The role of mucin-type O-glycans in eukaryotic development,” Seminars in Cell and Developmental Biology, vol. 21, no. 6, pp. 616–621, 2010.
[11]
G. W. Hart, “Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins,” Annual Review of Biochemistry, vol. 66, pp. 315–335, 1997.
[12]
T. Y. Chou, C. V. Dang, and G. W. Hart, “Glycosylation of the c-Myc transactivation domain,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 10, pp. 4417–4421, 1995.
[13]
W. H. Yang, J. E. Kim, H. W. Nam et al., “Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability,” Nature Cell Biology, vol. 8, no. 10, pp. 1074–1083, 2006.
[14]
G. W. Hart, M. P. Housley, and C. Slawson, “Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins,” Nature, vol. 446, no. 7139, pp. 1017–1022, 2007.
[15]
C. R. Torres and G. W. Hart, “Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc,” Journal of Biological Chemistry, vol. 259, no. 5, pp. 3308–3317, 1984.
[16]
C. F. Chou, A. J. Smith, and M. B. Omary, “Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18,” Journal of Biological Chemistry, vol. 267, no. 6, pp. 3901–3906, 1992.
[17]
W. G. Kelly, M. E. Dahmus, and G. W. Hart, “RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc,” Journal of Biological Chemistry, vol. 268, no. 14, pp. 10416–10424, 1993.
[18]
I. Han, E. S. Oh, and J. E. Kudlow, “Responsiveness of the state of O-linked N-acetylglucosamine modification of nuclear pore protein p62 to the extracellular glucose concentration,” Biochemical Journal, vol. 350, no. 1, pp. 109–114, 2000.
[19]
L. Wells, K. Vosseller, and G. W. Hart, “Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc,” Science, vol. 291, no. 5512, pp. 2376–2378, 2001.
[20]
D. C. Love and J. A. Hanover, “The hexosamine signaling pathway: deciphering the "O-GlcNAc code",” Science's STKE, vol. 2005, no. 312, p. re13, 2005.
[21]
N. E. Zachara and G. W. Hart, “Cell signaling, the essential role of O-GlcNAc!,” Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, vol. 1761, no. 5-6, pp. 599–617, 2006.
[22]
J. T. Clarke, “Simplified "Disc" (Polyaceylamide Gel) electrophoresis,” Annals of the New York Academy of Sciences, vol. 121, no. 2, pp. 428–436, 1964.
[23]
R. M. Zacharius, T. E. Zell, J. H. Morrison, and J. J. Woodlock, “Glycoprotein staining following electrophoresis on acrylamide gels,” Analytical Biochemistry, vol. 30, no. 1, pp. 148–152, 1969.
[24]
T. H. Steinberg, “Chapter 31 protein Gel staining methods. An introduction and overview,” Methods in Enzymology, vol. 463, no. C, pp. 541–563, 2009.
[25]
C. Hart, B. Schulenberg, T. H. Steinberg, W. Y. Leung, and W. F. Patton, “Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain,” Electrophoresis, vol. 24, no. 4, pp. 588–598, 2003.
[26]
A. H. Wardi and G. A. Michos, “Alcian blue staining of glycoproteins in acrylamide disc electrophoresis,” Analytical Biochemistry, vol. 49, no. 2, pp. 607–609, 1972.
[27]
M. R. Green, J. V. Pastewka, and A. C. Peacock, “Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye,” Analytical Biochemistry, vol. 56, no. 1, pp. 43–51, 1973.
[28]
H. Lis and N. Sharon, “Lectins as molecules and as tools,” Annual Review of Biochemistry, vol. 55, pp. 35–67, 1986.
[29]
T. Sata, C. Zuber, and J. Roth, “Lectin-digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry,” Histochemistry, vol. 94, no. 1, pp. 1–11, 1990.
[30]
P. Gravel, O. Golaz, C. Walzer, D. F. Hochstrasser, H. Turler, and L. P. Balant, “Analysis of glycoproteins separated by two-dimensional gel electrophoresis using lectin blotting revealed by chemiluminescence,” Analytical Biochemistry, vol. 221, no. 1, pp. 66–71, 1994.
[31]
F. I. Comer, K. Vosseller, L. Wells, M. A. Accavitti, and G. W. Hart, “Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine,” Analytical Biochemistry, vol. 293, no. 2, pp. 169–177, 2001.
[32]
G. D. Holt, C. M. Snow, and A. Senior, “Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine,” Journal of Cell Biology, vol. 104, no. 5, pp. 1157–1164, 1987.
[33]
C. M. Snow, A. Senior, and L. Gerace, “Monoclonal antibodies identify a group of nuclear pore complex glycoproteins,” Journal of Cell Biology, vol. 104, no. 5, pp. 1143–1156, 1987.
[34]
R. A. O'Neill, “Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis,” Journal of Chromatography A, vol. 720, no. 1-2, pp. 201–215, 1996.
[35]
V. Tretter, F. Altmann, and L. Marz, “Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1→3 to the asparagine-linked N-acetylglucosamine residue,” European Journal of Biochemistry, vol. 199, no. 3, pp. 647–652, 1991.
[36]
R. B. Trimble and A. L. Tarentino, “Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2, and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans,” Journal of Biological Chemistry, vol. 266, no. 3, pp. 1646–1651, 1991.
[37]
D. M. Carlson, “Structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins,” Journal of Biological Chemistry, vol. 243, no. 3, pp. 616–626, 1968.
[38]
T. Patel, J. Bruce, A. Merry et al., “Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins,” Biochemistry, vol. 32, no. 2, pp. 679–693, 1993.
[39]
N. H. Packer, M. A. Lawson, D. R. Jardine, and J. W. Redmond, “A general approach to desalting oligosaccharides released from glycoproteins,” Glycoconjugate Journal, vol. 15, no. 8, pp. 737–747, 1998.
[40]
K. O. Bornsen, M. D. Mohr, and H. M. Widmer, “Ion-exchange and purification of carbohydrates on a Nafion membrane as a new sample pretreatment for matrix-assisted laser-desorption ionization mass-spectrometry,” Rapid Communications in Mass Spectrometry, vol. 9, no. 11, pp. 1031–1034, 1995.
[41]
Y. Mechref and M. V. Novotny, “Structural investigations of glycoconjugates at high sensitivity,” Chemical Reviews, vol. 102, no. 2, pp. 321–369, 2002.
[42]
J. C. Bigge, T. P. Patel, J. A. Bruce, P. N. Goulding, S. M. Charles, and R. B. Parekh, “Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid,” Analytical Biochemistry, vol. 230, no. 2, pp. 229–238, 1995.
[43]
G. R. Guile, P. M. Rudd, D. R. Wing, S. B. Prime, and R. A. Dwek, “A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles,” Analytical Biochemistry, vol. 240, no. 2, pp. 210–226, 1996.
[44]
G. R. Guile, S. Y. C. Wong, and R. A. Dwek, “Analytical and preparative separation of anionic oligosaccharides by weak anion-exchange high-performance liquid chromatography on an inert polymer column,” Analytical Biochemistry, vol. 222, no. 1, pp. 231–235, 1994.
[45]
R. R. Townsend and M. R. Hardy, “Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography,” Glycobiology, vol. 1, no. 2, pp. 139–147, 1991.
[46]
M. P. Campbell, L. Royle, C. M. Radcliffe, R. A. Dwek, and P. M. Rudd, “GlycoBase and autoGU: tools for HPLC-based glycan analysis,” Bioinformatics, vol. 24, no. 9, pp. 1214–1216, 2008.
[47]
L. Royle, T. S. Mattu, E. Hart et al., “An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins,” Analytical Biochemistry, vol. 304, no. 1, pp. 70–90, 2002.
[48]
G. R. Guile, D. J. Harvey, N. O'Donnell et al., “Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland,” European Journal of Biochemistry, vol. 258, no. 2, pp. 623–656, 1998.
[49]
C. J. Edge, T. W. Rademacher, M. R. Wormald et al., “Fast sequencing of oligosaccharides: the reagent-array analysis method,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 14, pp. 6338–6342, 1992.
[50]
D. ？agi, P. Kienz, J. Denecke, T. Marquardt, and J. Peter-Katalini？, “Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation,” Proteomics, vol. 5, no. 10, pp. 2689–2701, 2005.
[51]
Y. Mechref and M. V. Novotny, “Structural investigations of glycoconjugates at high sensitivity,” Chemical Reviews, vol. 102, no. 2, pp. 321–369, 2002.
[52]
I. Ciucanu and F. Kerek, “A simple and rapid method for the permethylation of carbohydrates,” Carbohydrate Research, vol. 131, no. 2, pp. 209–217, 1984.
[53]
J. Jang-Lee, S. J. North, M. Sutton-Smith et al., “Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies,” Methods in Enzymology, vol. 415, pp. 59–86, 2006.
[54]
G. Alvarez-Manilla, N. L. Warren, T. Abney et al., “Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I,” Glycobiology, vol. 17, no. 7, pp. 677–687, 2007.
[55]
A. Dell, “F.A.B.-Mass Spectrometry of Carbohydrates,” Advances in Carbohydrate Chemistry and Biochemistry, vol. 45, no. C, pp. 19–72, 1987.
[56]
D. J. Harvey, R. H. Bateman, and M. R. Green, “High-energy collision-induced fragmentation of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry,” Journal of Mass Spectrometry, vol. 32, no. 2, pp. 167–187, 1997.
[57]
Z. M. Segu and Y. Mechref, “Characterizing protein glycosylation sites through higher-energy C-trap dissociation,” Rapid Communications in Mass Spectrometry, vol. 24, no. 9, pp. 1217–1225, 2010.
[58]
N. E. Scott, B. L. Parker, A. M. Connolly et al., “Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni,” Molecular and Cellular Proteomics, vol. 10, no. 2, 2011.
[59]
J. M. Prien, D. J. Ashline, A. J. Lapadula, H. Zhang, and V. N. Reinhold, “The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS,” Journal of the American Society for Mass Spectrometry, vol. 20, no. 4, pp. 539–556, 2009.
[60]
B. Küster, S. F. Wheeler, A. P. Hunter, R. A. Dwek, and D. J. Harvey, “Sequencing of N-linked oligosaccharides directly from protein gels: in- gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography,” Analytical Biochemistry, vol. 250, no. 1, pp. 82–101, 1997.
[61]
Z. Roth, S. Parnes, S. Wiel et al., “N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites,” Glycoconjugate Journal, vol. 27, no. 1, pp. 159–169, 2010.
[62]
B. Küster and M. Mann, “O-18-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching,” Analytical Chemistry, vol. 71, no. 7, pp. 1431–1440, 1999.
[63]
H. Hwang, J. Zhaang, K. A. Chung et al., “Glycoproteomics in neurodegenerative diseases,” Mass Spectrometry Reviews, vol. 29, no. 1, pp. 79–125, 2010.
[64]
A. M. Mayampurath, Y. Wu, Z. M. Segu, Y. Mechref, and H. Tang, “Improving confidence in detection and characterization of protein n-glycosylation sites and microheterogeneity,” Rapid Communications in Mass Spectrometry, vol. 25, no. 14, pp. 2007–2019, 2011.
[65]
S. A. Whelan and G. W. Hart, “Proteomic approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation,” Circulation Research, vol. 93, no. 11, pp. 1047–1058, 2003.
[66]
K. D. Greis, B. K. Hayes, F. I. Comer et al., “Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by β-elimination and tandem electrospray mass spectrometry,” Analytical Biochemistry, vol. 234, no. 1, pp. 38–49, 1996.
[67]
S. P. Gygi, B. Rist, S. A. Gerber, F. Turecek, M. H. Gelb, and R. Aebersold, “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags,” Nature Biotechnology, vol. 17, no. 10, pp. 994–999, 1999.
[68]
P. L. Ross, Y. N. Huang, J. N. Marchese et al., “Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents,” Molecular and Cellular Proteomics, vol. 3, no. 12, pp. 1154–1169, 2004.
