Abstract
Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.
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Baenziger, J. U.; Kuma, S.; Brodbeck, R. M.; Smith, P. L.; Beranek, M. C. Circulatory Half-Life but not Interaction with the Lutropin/Chorionic Gonadotropin Receptor is Modulated by Sulfation of Bovine Lutropin Oligosaccharides. Proc. Nat. Acad. Sci. U.S.A. 1992, 89(1), 334–338.
Baenziger, J. U.; Green, E. D. Pituitary Glycoprotein Hormone Oligosaccharides: Structure, Synthesis, and Function of the Asparagine-Linked Oligosaccharides on Lutropin, Follitropin, and Thyrotropin. Biochim. Biophys. Acta Rev. Biomembr. 1988, 947(2), 287–306.
Stockell Hartree, A.; Renwick, A. G. Molecular Structures of Glycoprotein Hormones and Functions of Their Carbohydrate Components. Biochem. J. 1992, 287(3), 665–679.
lmai, Y.; Lasky, L. A.; Rosen, S. D. Sulphation Requirement for GlyCAM-1, an Endothelial Ligand for L-Selectin. Nature 1993, 361(6412), 555–557.
Imai, Y.; Singer, M. S.; Fennie, C.; Lasky, L. A.; Rosen, S. D. Identification of a Carbohydrate-Based Endothelial Ligand for a Lymphocyte Homing Receptor. J. Cell Biol. 1991, 113(5), 1213–1221.
Hemmerich, S.; Bertozzi, C. R.; Leffler, H.; Rosen, S. D. Identification of the Sulfated Monosaccharides of GlyCAM-1, an Endothelial-Derived Ligand for L-Selectin. Biochemistry 1994, 33(16), 4820–4829.
Shilatifard, A.; Merkle, R. K.; Helland, D. E.; Welles, J. L.; Haseltine, W. A.; Cummings, R. D. Complex-Type N-Linked Oligosaccharides of gp120 from Human Immunodeficiency Virus Type 1 Contain Sulfated N-Acetylglucosamine. J. Virol. 1993, 67(2), 943–952.
Fukuda, M.; Hiraoka, N.; Akama, T. O.; Fukud, M. N. Carbohydratemodifying Sulfotransferases: Structure, Function, and Pathophysiology. J. Biol. Chem. 2001, 276.
Martini, R.; Xin, Y.; Schmitz, B.; Schachner, M. The L2/HNK-1 Carbohydrate Epitope Is Involved in the Preferential Outgrowth of Motor Neurons on Ventral Roots and Motor Nerves. Eur. J. Neurosci. 1992, 4(7), 628–639.
Chance, D. L.; Mawhinney, T. P. Disulfated Oligosaccharides Derived from Tracheobronchial Mucous Glycoproteins of a Patient Suffering from Cystic Fibrosis. Carbohydr. Res. 1996, 295, 157–177.
Fuster, M. M.; Esko, J. D. The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets. Nat. Rev. Cancer 2005, 5(7), 526–542.
Xiong, L.; Andrews, D.; Regnier, F. Comparative Proteomics of Glycoproteins Based on Lectin Selection and Isotope Coding. J. Proteome Res. 2003, 2(6), 618–625.
Thomsson, K. A.; Karlsson, N. G.; Hansson, G. C. Liquid Chromatography-Electrospray Mass Spectrometry as a Tool for the Analysis of Sulfated Oligosaccharides from Mucin Glycoproteins. J. Chromatogr. A 1999, 854(1/2), 131–139.
Ohtsubo, K.; Marth, J. D. Glycosylation in Cellular Mechanisms of Health and Disease. Cell 2006, 125, 855–867.
Freeze, H.; Wolgast, D. Structural Analysis of N-Linked Oligosaccharides from Glycoproteins Secreted by Dictyostelium discoideum: Identification of Mannose 6-sulfate. J. Biol. Chem. 1986, 261(1), 127–134.
Bowman, K. G.; Cook, B. N.; de Graffenried, C. L.; Bertozzi, C. R. Biosynthesis of L-Selectin Ligands: Sulfation of Sialyl Lewis x-Related Oligosaccharides by a Family of GlcNAc-6-Sulfotransferases. Biochemistry 2001, 40(18), 5382–5391.
Hard, K.; Van Zadelhoff, G.; Moonen, P.; Kamerling, J. P.; Vliegenthart, J. F. G. The Asn-Linked Carbohydrate Chains of Human Tamm-Horsfall Glycoprotein of One Male. Eur. J. Biochem. 1992, 209, 895–915.
van Rooijen, J. J. M.; Kamerling, J. P.; Vliegenthart, J. F. G. Sulfated Di-, Tri-, and Tetra-Antennary N-Glycans in Human Tamm-Horsfall Glycoprotein. Eur. J. Biochem. 1998, 256(2), 471–487.
Zaia, J. Mass Spectrometry of Oligosaccharides. Mass Spectrom. Rev. 2004, 23, 161–227.
Mechref, Y.; Novotny, M. V. Miniaturized Separation: Techniques in Glycomic Investigations. J. Chromatogr. B 2006, 841, 65–78.
Mechref, Y.; Novotny, M. V. Structural Investigations of Glycoconjugates at High Sensitivity. Chem. Rev. 2002, 102, 321–370.
Jiang, H.; Irungu, J.; Desaire, H. Enhanced Detection of Sulfated Glycosylation Sites in Glycoproteins. J. Am. Soc. Mass Spectrom. 2005, 16(3), 340–348.
Irungu, J.; Dalpathado, D. S.; Go, E. P.; Jiang, H.; Ha, H.-V.; Bousfield, G. R.; Desaire, H. Method for Characterizing Sulfated Glycoproteins in a Glycosylation Site-Specific Fashion, Using Ion Pairing and Tandem Mass Spectrometry. Anal. Chem. 2006, 78(4), 1181–1190.
Imami, K.; Ishihama, Y.; Terabe, S. On-line Selective Enrichment and Ion-pair Reaction for Structural Determination of Sulfated Glycopeptides by Capillary Electrophoresis-Mass Spectrometry. J. Chromatogr. A 2008, 1194(2), 237–242.
Barboza, M.; Duschak, V. G.; Fukuyama, Y.; Nonami, H.; Erra-Balsells, R.; Cazzulo, J. J.; Couto, A. S. Structural Analysis of the N-glycans of the Major Cysteine Proteinase of Trypanosoma cruzi. FEBS J. 2005, 272, 3803–3815.
Murakami, T.; Natsuka, S.; Nakakita, S.-I.; Hase, S. Structure Determination of a Sulfated N-Glycans, Candidate for a Precursor of the Selectin Ligand in Bovine Lung. Glycoconj. J. 2007, 24(4), 195–206.
Lo-Guidice, J.-M.; Herz, H.; Lamblin, G.; Plancke, Y.; Roussel, P.; Lhermitte, M. Structures of Sulfated Oligosaccharides Isolated from the Respiratory Mucins of a Nonsecretor (O, Lea+b-) Patient Suffering from Chronic Bronchitis. Glycoconj. J. 1997, 14(1), 113–125.
Yagi, H.; Takahashi, N.; Yamaguchi, Y.; Kimura, N.; Uchimura, K.; Kannagi, R.; Kato, K. Development of Structural Analysis of Sulfated N-Glycans by Multidimensional High Performance Liquid Chromatography Mapping Methods. Glycobiol. 2005, 15, 1051–1060.
Hermentin, P.; Witzel, R.; Doenges, R.; Bauer, R.; Haupt, H.; Patel, T.; Parekh, R. B.; Brazel, D. The Mapping by High-pH Anion-Exchange Chromatography with Pulsed Amperometric Detection and Capillary Electrophoresis of the Carbohydrate Moieties of Human Plasma [α]1-acid Glycoprotein. Anal. Biochem. 1992, 206(2), 419–429.
Kang, P.; Mechref, Y.; Novotny, M. V. High-Throughput Solid-phase Permethylation of Glycans Prior to Mass Spectrometry. Rapid Commun. Mass Spectrom. 2008, 22(5), 721–734.
Kang, P.; Mechref, Y.; Klouckova, I.; Novotny, M. V. Solid-Phase Permethylation of Glycans for Mass Spectrometric Analysis. Rapid Commun. Mass Spectrom. 2005, 19, 3421–3428.
Kopaciewicz, W.; Regnier, F. E. Mobile Phase Selection for the High-Performance Ion-Exchange Chromatography of Proteins. Anal. Biochem. 1983, 133(1), 251–259.
Wheeler, S. F.; Harvey, D. J. Extension of the In-Gel Release Method for Structural Analysis of Neutral and Sialylated N-Linked Glycans to the Analysis of Sulfated Glycans: Application to the Glycans from Bovine Thyroid-Stimulating Hormone. Anal. Biochem. 2001, 296(1), 92–100.
Morelle, W.; Donadio, S.; Ronin, C.; Michalski, J.-C. Characterization of N-Glycans of Recombinant Human Thyrotropin using Mass Spectrometry. Rapid Commun. Mass Spectrom. 2006, 20(3), 331–345.
Mechref, Y.; Novotny, M. V. Mass Spectrometric Mapping and Sequencing of N-Linked Oligosaccharides Derived from Submicrogram Amounts of Glycoproteins. Anal. Chem. 1998, 70(3), 455–463.
Devakumar, A.; Mechref, Y.; Kang, P.; Novotny, M. V.; Reilly, J. P. Laser-Induced Photofragmentation of Neutral and Acidic Glycans Inside an Ion-Trap Mass Spectrometer. Rapid Commun. Mass Spectrom. 2007, 21(8), 1452–1460.
Ciucanu, I.; Costello, C. E. Elimination of Oxidative Degradation during the Per-O-Methylation of Carbohydrates. J. Am. Chem. Soc. 2003, 125, 16213–16219.
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Published online September 30, 2009
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Lei, M., Novotny, M.V. & Mechref, Y. Sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements. J Am Soc Mass Spectrom 21, 348–357 (2010). https://doi.org/10.1016/j.jasms.2009.09.017
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DOI: https://doi.org/10.1016/j.jasms.2009.09.017