Abstract
This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHα (ovine luteinizing hormone, α-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn56 site of oLHα. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn56 site of oLHα was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.
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References
Harvey, D. J. Identification of Protein-Bound Carbohydrates by Mass Spectrometry. Proteomics 2001, 1, 311–328.
Rouse, J. C.; Vath, J. E. On-the-Probe Sample Cleanup Strategies for Glycoprotein-Released Carbohydrates Prior to Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Spectrometry. Anal. Biochem. 1996, 238, 82–92.
Scanlan, C. N.; Pantophlet, R.; Wormald, M. R.; Saphire, E. O.; Stanfield, R.; Wilson, I. A.; Katinger, H.; Dwek, R. A.; Rudd, P. M.; Burton, D. R. The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α→2 Mannose Residues on the Outer Face of gp120. J. Virol. 2002, 76, 7306–7321.
Harvey, D. J. N-(2-Diethylamino)Ethyl-4-Aminobenzamide Derivative for High Sensitivity Mass Spectrometric Detection and Structure Determination of N-Linked Carbohydrates. Rapid Commun. Mass Spectrom. 2000, 14, 862–871.
Mechref, Y.; Novotny, M. V. Structural Investigations of Glycoconjugates at High Sensitivity. Chem. Rev. 2002, 102, 321–369.
Huddleston, M. J.; Bean, M. F.; Carr, S. A. Collisional Fragmentation of Glycopeptides by Electrospray Ionization LC/MS and LC/MS/MS: Methods for Selective Detection of Glycopeptides in Protein Digests. Anal. Chem. 1993, 65, 877–884.
Juhasz, P.; Martin, S. A. The Utility of Nonspecific Proteases in the Characterization of Glycoproteins by High-Resolution Time-of-Flight Mass Spectrometry. Int. J. Mass Spectrom. 1997, 169/170, 217–230.
Zhu, X.; Borchers, C.; Bienstock, R. J.; Tomer, K. B. Mass Spectrometric Characterization of Glycosylation Pattern of HIV-gp120 Expressed in CHO Cells. Biochem 2000, 39, 11194–11204.
Dell, A.; Morris, H. R. Glycoprotein Structure Determination by Mass Spectrometry. Science 2001, 291, 2351–2356.
An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Determination of N-Glycosylation Sites and Site Heterogeneity in Glycoproteins. Anal. Chem. 2003, 75, 5628–5673.
Carr, S. A.; Huddleston, M. J.; Bean, M. F. Selective Identification and Differentiation of N- and O-Linked Oligosaccharides in Glycoproteins by Liquid Chromatography-Mass Spectrometry. Protein Sci 1993, 2, 183–196.
Ma, S.; Hill, K. E.; Burk, R. F.; Caprioli, R. M. Mass Spectrometric Identification of N- and O-Glycosylation Sites of Full-Length Rat Selenoprotein P and Determination of Selenide-Sulfide and Disulfide Linkages in the Shortest Isoform. Biochem 2003, 42, 9703–9711.
Zhen, Y.; Caprioli, R. M.; Staros, J. V. Characterization of Glycosylation Sites of the Epidermal Growth Factor Receptor. Biochem 2003, 42, 5478–5492.
Matzuk, M. M.; Boime, I. Site-Specific Mutagenesis Defines the Intracellular Role of the Asparagine-Linked Oligosaccharides of Chrionic Gonadotropin β-Subunit. J. Biol. Chem. 1988, 263, 17106–17111.
Butnev, V. Y.; Gotschall, R. R.; Butnev, V. Y.; Baker, V. L.; Moore, W. T.; Bousfield, G. R. Hormone-Specific Inhibitory Influence of α-Subunit Asn56 Oligosaccharide on in Vitro Subunit Association and FSH Receptor Binding of Equine Gonadotropins. Biol. Repro. 1998, 58, 458–469.
Matsuk, M. M.; Keene, J. L.; Boime, I. Site Specificity of the Chorionic Gonadotropin N-Linked Oligosaccharides in Signal Transduction. J. Biol. Chem. 1989, 264, 2409–2414.
Liu, W.-K.; Ascoli, M.; Ward, D. N. Ovine Lutropin Subunit Isolation: Comparison of Salt Precipitation and Countercurrent Distribution Procedures. J. Biol. Chem. 1977, 252, 5274–5279.
Bousfield, G. R.; Baker, V. L.; Gotschall, R. R.; Butnev, V. Y. Carbohydrate Analysis of Glycoprotein Hormones. Methods 2000, 21, 15–39.
Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. A General Approach to Desalting Oligosaccharides Released from Glycoproteins. Glycoconj. J. 1998, 15, 737–747.
Weisshaar, G.; Hiyama, J.; Renwick, A. G. C. Site-Specific N-Glycosylation of Ovine Lutropin. Structural Analysis of One- and Two-Dimensional Proton NMR Spectroscopy. Eur. J. Biochem. 1990, 192(741), 751.
Thotakura, N. R.; Blithe, D. L. Glycoprotein Hormones: Glycobiology of Gonadotrophins, Thyrotrophin, and Free α-Subunit. Glycobiology 1995, 5, 3–10.
Ritchie, M. A.; Gill, A. C.; Deery, M. J.; Lilley, K. Precursor Ion Scanning for Detection and Structural Characterization of Heterogeneous Glycopeptide Mixtures. J. Am. Soc. Mass Spectrom. 2002, 13, 1065–1077.
Yagami, T.; Kitagawa, K.; Futaki, S. Analysis of Sulfated Tyrosine-Containing Peptides by Liquid Secondary-Ion Mass Spectrometry with Constant Neutral-Loss (80 amu) scanning. Anal. Sci. 1995, 11, 1025–1028.
Hakansson, K.; Emmett, M. R.; Marshall, A. G.; Davidsson, P.; Nilsson, C. L. Structural Analysis of 2D-Gel-Separated Glycoproteins from Human Cerebrospinal Fluid by Tandem High-Resolution Mass Spectrometry. J. Proteome Res. 2003, 2(6), 581–588.
Liu, T.; Li, J. D.; Zeng, R.; Shao, X. X.; Wang, K. Y.; Xia, Q. C. Capillary Electrophoresis-Electrospray Mass Spectrometry for the Characterization of High-Mannose-Type N-Glycosylation and Differential Oxidation in Glycoproteins by Charge Reversal and Protease/Glycosidase Digestion. Anal. Chem. 2001, 73, 5875–5885.
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Published online March 12, 2004
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Jiang, H., Desaire, H., Butnev, V.Y. et al. Glycoprotein profiling by electrospray mass spectrometry. J Am Soc Mass Spectrom 15, 750–758 (2004). https://doi.org/10.1016/j.jasms.2004.01.009
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DOI: https://doi.org/10.1016/j.jasms.2004.01.009