Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 1: Methodology)

  • Anja Pfenninger
  • Michael KarasEmail author
  • Berndt Finke
  • Bernd Stahl


Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M − H] with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a “new” reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MSn capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal β1→3 GlcNAc or Gal β1→4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.


Oligosaccharide GlcNAc Fucose Collision Induce Dissociation Mass Increment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Dwek, R. A. Chem. Rev. 1996, 96, 683.CrossRefGoogle Scholar
  2. 2.
    Dell, A. Methods Enzymol. 1990, 193, 647.CrossRefGoogle Scholar
  3. 3.
    Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1997, 6, 331.CrossRefGoogle Scholar
  4. 4.
    Settineri, C. A.; Burlingame, A. L. Carbohydrate Analysis; Journal of Chromatography Library. Elsevier: Amsterdam, 1995; p 447.Google Scholar
  5. 5.
    Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349.CrossRefGoogle Scholar
  6. 6.
    Domon, B.; Costello, C. E. Glycoconj. J. 1988, 5, 397.CrossRefGoogle Scholar
  7. 7.
    Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991, 63, 1463.CrossRefGoogle Scholar
  8. 8.
    Spengler, B. J. Mass Spectrom. 1997, 32, 1019.CrossRefGoogle Scholar
  9. 9.
    Harvey, D. J.; Küster, B.; Naven, T. J. P. Glycoconj. J. 1998, 15, 333.CrossRefGoogle Scholar
  10. 10.
    Spengler, B.; Kirsch, D.; Kaufmann, R.; Lemoine, J. J. Org. Mass Spectrom. 1994, 29, 782.CrossRefGoogle Scholar
  11. 11.
    Lemoine, J.; Chirat, F.; Domon, B. J. Mass Spectrom. 1996, 31, 908.CrossRefGoogle Scholar
  12. 12.
    Harvey, D. J.; Naven, T. J. P.; Küster, B.; Bateman, R. H.; Green, M.; Critchley, G. Rapid. Commun. Mass Spectrom. 1995, 9, 1556.CrossRefGoogle Scholar
  13. 13.
    Okamoto, M.; Takahashi, K.; Doi, T.; Takimoto, Y. Anal. Chem. 1997, 69, 2919.CrossRefGoogle Scholar
  14. 14.
    Talbo, G.; Mann, M. Rapid Commun. Mass Spectrom. 1996, 10, 100.CrossRefGoogle Scholar
  15. 15.
    Viseux, N.; Costello, C. E.; Domon, B. J. Mass Spectrom. 1999, 34, 364.CrossRefGoogle Scholar
  16. 16.
    Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B.; Sawatzki, G. J. Mass Spectrom. 1999, 34, 99.CrossRefGoogle Scholar
  17. 17.
    Naven, T. J. P.; Harvey, D. J.; Brown, J.; Critchley, G. Rapid Commun. Mass Spectrom. 1997, 11, 1681.CrossRefGoogle Scholar
  18. 18.
    Spina, E.; Cozzolino, R.; Ryan, E.; Garozzo, D. J. Mass. Spectrom. 2000, 35, 1042.CrossRefGoogle Scholar
  19. 19.
    Sutton, C. W.; O’Neill, J. A.; Cottrell, J. S. Anal. Biochem. 1994, 218, 34.CrossRefGoogle Scholar
  20. 20.
    Küster, B.; Naven, T. J. P.; Harvey, D. J. J. Mass Spectrom. 1996, 31, 1131.CrossRefGoogle Scholar
  21. 21.
    Yang, Y.; Orlando, R. Anal. Chem. 1996, 68, 570.CrossRefGoogle Scholar
  22. 22.
    Mechref, Y.; Novotny, M. V. Anal. Chem. 1998, 70, 455.CrossRefGoogle Scholar
  23. 23.
    Geyer, H.; Schmitt, S.; Wuhrer, M.; Geyer, R. Anal. Chem. 1999, 71, 476.CrossRefGoogle Scholar
  24. 24.
    Cancilla, M. T.; Penn, S. G.; Lebrilla, C. B. Anal. Chem. 1998, 70, 663.CrossRefGoogle Scholar
  25. 25.
    Wong, A. W.; Cancilla, M. T.; Voss, L. R.; Lebrilla, C. B. Anal. Chem. 1999, 71, 205.CrossRefGoogle Scholar
  26. 26.
    Hayes, R. N.; Gross, M. L. Methods Enzymol. 1990, 193, 237.CrossRefGoogle Scholar
  27. 27.
    Yost, R. A.; Boyd, R. K. Methods Enzymol. 1990, 193, 154.CrossRefGoogle Scholar
  28. 28.
    Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772.CrossRefGoogle Scholar
  29. 29.
    Sheeley, D. M.; Reinhold, V. N. Anal. Chem. 1998, 70, 3053.CrossRefGoogle Scholar
  30. 30.
    Reinhold, V. N.; Reinhold, B. B.; Chan, S. Methods Enzymol. 1996, 271, 377.CrossRefGoogle Scholar
  31. 31.
    Souluki, T.; Reinhold, B. B.; Costello, C. E.; O’Malley, M.; Guan, S.; Marshall, A. G. Anal. Chem. 1998, 70, 857.CrossRefGoogle Scholar
  32. 32.
    Fura, A.; Leary, J. A. Anal. Chem. 1993, 65, 2805.CrossRefGoogle Scholar
  33. 33.
    Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293.CrossRefGoogle Scholar
  34. 34.
    Smith, G.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1996, 7, 953.CrossRefGoogle Scholar
  35. 35.
    König, S.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1998, 9, 1125.CrossRefGoogle Scholar
  36. 36.
    Viseux, N.; de Hoffmann, E.; Domon, B. Anal. Chem. 1998, 70, 4951.CrossRefGoogle Scholar
  37. 37.
    Spengler, B.; Dolce, J. W.; Cotter, R. J. Anal. Chem. 1990, 62, 1731.CrossRefGoogle Scholar
  38. 38.
    Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964.CrossRefGoogle Scholar
  39. 39.
    Domon, B.; Müller, D. R.; Richter, W. J. Org. Mass Spectrom. 1989, 24, 357.CrossRefGoogle Scholar
  40. 40.
    Smith, G.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1996, 7, 953.CrossRefGoogle Scholar
  41. 41.
    Bahr, U.; Pfenninger, A.; Karas, M.; Stahl, B. Anal. Chem. 1997, 69, 4530.CrossRefGoogle Scholar
  42. 42.
    Asam, G. L.; Glish, G. J. Am. Soc. Mass Spectrom. 1997, 8, 987.CrossRefGoogle Scholar
  43. 43.
    Mulroney, B.; Traeger, J. C.; Stone, B. A. J. Mass Spectrom. 1995, 30, 1277.CrossRefGoogle Scholar
  44. 44.
    Garozzo, G.; Impallomeni, E.; Spina, E.; Green, B. N.; Hutton, T. Carbohydr. Res. 1991, 221, 1283.CrossRefGoogle Scholar
  45. 45.
    Bahr, U.; Karas, M.; Schubert-Zsilavecz, M. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, May, 1998; p 2.Google Scholar
  46. 46.
    Friedl, C. H.; Lochnit, G.; Geyer, R.; Karas, M.; Bahr, U. Anal. Biochem. 2000, 284, 279.CrossRefGoogle Scholar
  47. 47.
    Kunz, C.; Rudolf, S. Acta Paediatr. 1993, 82, 903.CrossRefGoogle Scholar
  48. 48.
    Kobata, A.; Yamashita, K.; Tachibana, Y. Methods Enzymol. 1978, 50, 216.CrossRefGoogle Scholar
  49. 49.
    Kuntz, R.; Dabrowski, U.; Dabrowski, J.; Ebersold, A.; Peter-Katalinic, J.; Egge, H. Biol. Chem. Hoppe-Seyler 1988, 369, 257.Google Scholar
  50. 50.
    Strecker, G.; Wieruszeski, J. M.; Michalksi, J. C.; Montreuil, J. Glycoconj. J. 1989, 6, 169.CrossRefGoogle Scholar
  51. 51.
    Stahl, B.; Thurl, S.; Zeng, J.; Karas, M.; Hillenkamp, F.; Steup, M.; Sawatzki, G. Anal. Biochem. 1994, 223, 218.CrossRefGoogle Scholar
  52. 52.
    Finke, B.; Stahl, B.; Pfenninger, A.; Karas, M.; Daniel, H.; Sawatzki, G. Anal. Chem. 1999, 71, 3755.CrossRefGoogle Scholar
  53. 53.
    Thurl, S.; Offermanns, J.; Müller-Werner, B.; Sawatzki, G. J. Chromatogr. 1991, 568, 291.CrossRefGoogle Scholar
  54. 54.
    Thurl, S.; Müller-Werner, B.; Sawatzki, G. Anal. Biochem. 1996, 235, 202.CrossRefGoogle Scholar
  55. 55.
    Chai, W.; Piskarev, V.; Lawson, A. M. Anal. Chem. 2001, 73, 651.CrossRefGoogle Scholar
  56. 56.
    Bier, M. E.; Schwartz, J. C. In Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; p 235.Google Scholar
  57. 57.
    Kennedy, J. F. Carbohydrate Chemistry. Clarendon Press: Oxford, 1988, 269.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2002

Authors and Affiliations

  • Anja Pfenninger
    • 1
    • 3
  • Michael Karas
    • 1
    Email author
  • Berndt Finke
    • 2
  • Bernd Stahl
    • 2
  1. 1.Institute for Pharmaceutical Chemistry, BiocenterJ. W. Goethe University FrankfurtFrankfurtGermany
  2. 2.Numico Research Group GermanyFriedrichsdorf/TaunusGermany
  3. 3.Aventis Pharma Deutschland GmbHFrankfurt/MainGermany

Personalised recommendations