Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures


The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrPSc. These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex—Hexose, C6H12O6 carbohydrates, including mannnose and galactose; HexNAc—N-acetylhexosamine, C8H15NO6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc—N-acetylglucosamine; GalNAc—N-acetylgalactosamine; Fuc—Fucose; NeuAC—N-acetylneuraminic acid or sialic acid; TSE—Transmissible Spongiform Encephalopathy.]


  1. 1.

    Helenius, A.; Aebi, M. Intracellular Functions of N-Linked Glycans. Science 2001, 291, 2364–2369.

    CAS  Article  Google Scholar 

  2. 2.

    Dwek, R. A. Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 1996, 96, 683–720.

    CAS  Article  Google Scholar 

  3. 3.

    Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Glycosylation and the Immune System. Science 2001, 291, 2370–2376.

    CAS  Article  Google Scholar 

  4. 4.

    Parekh, R. B.; Dwek, R. A.; Sutton, B. J.; Fernandes, D. L.; Leung, A.; Stanworth, D.; Rademacher, T. W. Association of Rhuematoid Arthritis and Primary Osteoarthritis with Changes in the Glycosylation Pattern of Total Serum IgG. Nature 1985, 316, 452–457.

    CAS  Article  Google Scholar 

  5. 5.

    Parekh, R.; Isenberg, D.; Rook, G.; Roitt, A. Comparative Analysis of Disease-Associated Changes in the Galactosylation of Serum IgG. J. Autoimmun. 1989, 2, 101–114.

    CAS  Article  Google Scholar 

  6. 6.

    Turner, G. Haptoglobin—A Potential Reporter Molecule for Glycosylation Changes in Disease. Adv. Exp. Med. Biol. 1995, 376, 231–238.

    CAS  Google Scholar 

  7. 7.

    Maguire, T.; Breen, K. A Decrease in Neural Sialyltransferase Activity in Alzheimers Disease. Dementia 1995, 6, 185–90.

    CAS  Google Scholar 

  8. 8.

    Maguire, T.; Thakore, J.; Dinan, T. G.; Hopwood, S.; Breen, K. C. Plasma Sialyltransferase Levels in Psychiatric Disorders as a Possible Indicator of HPA Axis Function. Biol. Psychiat. 1997, 41, 1131–1136.

    CAS  Article  Google Scholar 

  9. 9.

    Mackiewicz, A.; Dewey, M. J.; Berger, F. G.; Baumann, H. Acute Phase Mediated Change in Glycosylation of Rat Alpha 1-Acid Glycoprotein in Transgenic Mice. Glycobiol. 1991, 1, 265–269.

    CAS  Article  Google Scholar 

  10. 10.

    Medzihradszky, K. F.; Maltby, D. A.; Hall, S. C.; Settineri, C. A.; Burlingame, A. L. Characterization of Protein N-Glycosylation by Reversed-Phase Microbore Liquid Chromatography/Electrospray Mass Spectrometry, Complementary Mobile Phases, and Sequential Exoglycosidase Digestion. Am. Soc. Mass Spectrom. 1994, 5, 350–358.

    CAS  Article  Google Scholar 

  11. 11.

    Wilm, M.; Neubauer, G.; Mann, M. Parent Ion Scans of Unsepparated Peptide Mixtures. Anal. Chem. 1996, 68, 527–533.

    CAS  Article  Google Scholar 

  12. 12.

    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.

    CAS  Article  Google Scholar 

  13. 13.

    Gerwig, G.; Vliegenthart, J. Analysis of Glycoprotein-Derived Glycopeptides. EXS 2000, 88, 159–186.

    CAS  Google Scholar 

  14. 14.

    Stimson, E.; Hope, J.; Chong, A.; Burlingame, A.L. Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions. Biochem. 1999, 38, 4885–4895.

    CAS  Article  Google Scholar 

  15. 15.

    Rudd, P. M.; Endo, T.; Colominas, C.; Groth, D.; Wheeler, S. F.; Harvey, D. J.; Wormwald, M. R.; Serban, H.; Prusiner, S. B.; Kobata, A.; Dwek, R. A. Glycosylation Differences Between the Normal and Pathogenic Prion Isoforms. PNAS U.S.A. 1999, 96, 13044–13049.

    CAS  Article  Google Scholar 

  16. 16.

    Hope, J.; Multhaup, G.; Reekie, L. J.; Kimberlin, R. H.; Beyreuther, K. Molecular Pathology of Scrapie-Associated Fibril Protein (PrP) in Mouse Brain Affected by the ME7 Strain of Scrapie. Eur. J. Biochem. 1988, 172, 271–277.

    CAS  Article  Google Scholar 

  17. 17.

    Annan, R. S.; Carr, S. A. The Essential Role of Mass Spectrometry in Characterizing Protein Structure: Mapping Posttranslational Modifications. J. Protein Chem. 1997, 16, 391–402.

    CAS  Article  Google Scholar 

  18. 18.

    Domon, B.; Costello, C.E. A Sytematic Nomenclature for Carbohydrate Fragmentations in FAB/MS Spectra of Glycocojugates. Glycoconj. 1988, 5, 397–409.

    CAS  Article  Google Scholar 

  19. 19.

    Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; Van Halbeek, H. The Asparagine Linked Oligosaccharides on Bovine Fetuin. J. Biol. Chem. 1988, 263, 18253–18268.

    CAS  Google Scholar 

  20. 20.

    Rice, K. G.; Rao, N. B. N.; Lee, Y. C. Large Scale Preparation and Characterization of N-Linked Glycopeptides from Bovine Fetuin. Anal. Biochem. 1990, 184, 249–258.

    CAS  Article  Google Scholar 

  21. 21.

    Stahl, N.; Baldwin, M. A.; Hecker, R.; Pan, K. M.; Burlingame, A. L.; Prusiner, S. B. Glycosylinositol Phospholipid Anchors of the Scrapie and Cellular Prion Proteins Contain Sialic-Acid. Biochem. 1992, 31, 5043–5053.

    CAS  Article  Google Scholar 

  22. 22.

    Turk, E.; Teplow, D. B.; Hood, L. E.; Prusiner, S. B. Purification and Properties of the Cellular and Scrapie Hamster Prion Proteins. Eur. J. Biochem. 1988, 176, 21–30.

    CAS  Article  Google Scholar 

  23. 23.

    Gill, A. C.; Ritchie, M. A.; Hunt, L. G.; Steane, S. E.; Davis, K. G.; Bocking, S. P.; Rhie, A. G. O.; Bennett, A. D.; Hope, J. Post-Translational Hydroxylation at the N-Terminus of the Prion Protein Reveals the Presence of PPII Structure in Vivo. EMBO J. 2000, 19, 5324–5331.

    CAS  Article  Google Scholar 

  24. 24.

    Lehmann, S.; Harris, D. A. Blockade of Glycosylation Promotes Acquisition of Scrapie-Like Properties by the Prion Protein in Cultured Cells. J. Biol. Chem. (1997, 272, 21479–21487)

    CAS  Article  Google Scholar 

  25. 25.

    Hope, J.; Wood, S.; Birkett, C.; Chong, A.; Bruce, M.; Cairns, D.; Goldmann, W.; Hunter, M.; Bostock, C. Molecular Analysis of Ovine Prion Protein Identifies Similarties Between BSE and an Experimental Isolate of Natural Scrapie, CH1641. J. Gen. Virol. 1999, 80, 1–4.

    CAS  Google Scholar 

  26. 26.

    Priola, S. A.; Lawson, V. A. Glycosylation Influences Cross-Species Formation of Protease-Resistant Prion Protein. EMBO 2001, 20, 3392–6699.

    Google Scholar 

  27. 27.

    Somerville, R. A. Host and Transmissible Spongiform Encephalopathy Agent Strain Control Glycosylation of PrP. J. Gen. Virol. 1999, 80, 1865–1872.

    CAS  Google Scholar 

  28. 28.

    Stahl, N.; Baldwin, M. A.; Teplow, D.; Hood, L.; Beavis, R.; Chait, B.; Gibson, B. W.; Burlingame, A. L.; Prusiner, S. B. Prion Diseases of Humans and Animals. Ellis Horwood Ltd: Chichester, UK, 1992; pp. 361–379.

    Google Scholar 

  29. 29.

    Chen, Y. J.; Wing, D. R.; Guile, G. R.; Dwek, R. A.; Harvey, D. J.; Zamze, S. Nuetral N-Glycans in Adult Rat Brain Tissue. Eur. J. Biochem. 1997, 251, 691–703.

    Article  Google Scholar 

  30. 30.

    Zamze, S.; Harvey, D. J.; Chen, Y. J.; Guile, G. R.; Dwek, R. A.; Wing, D. R. Sialylated N-Glycans in Adult Rat Brain Tissue—a Widespread Distribution of Disialylated Antennae in Complex and Hybrid Structures. Eur. J. Biochem. 1998, 258, 243–270.

    CAS  Article  Google Scholar 

  31. 31.

    Harvey, D. J. Electrospray Mass Spectrometry and Fragmentation of N-linked Carbohydrates Derivatized at the Reducing Terminus. J. Am. Soc. Mass Spectrom. 2000, 11, 900–915.

    CAS  Article  Google Scholar 

  32. 32.

    Spina, E.; Cozzolino, R.; Ryan, E.; Garozzo, D. Sequencing of Oligosaccharides by Collision Induced Dissociation Matrix Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass. Spectrom. 2000, 35, 1042–1048.

    CAS  Article  Google Scholar 

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Correspondence to Andrew C. Gill.

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Ritchie, M.A., Gill, A.C., Deery, M.J. et al. Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures. J. Am. Soc. Spectrom. 13, 1065–1077 (2002).

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  • Sialic Acid
  • GlcNAc
  • Glycopeptide
  • Prion Protein
  • Bovine Spongiform Encephalopathy