Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

  • Bruce Onisko
  • Irina Dynin
  • Jesús R. Requena
  • Christopher J. Silva
  • Melissa Erickson
  • John Mark Carter


Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein. Detection of VVEQMCTTQYQK was more sensitive than for the derivative with phenylisothiocyanate (PITC) because of decreased ionization efficiency of the PITC-derivatized peptides. The VVEQMCTTQYQK method has a LOD of 20 to 30 amol for pure standards. Proof of principle is demonstrated by quantitation of the amount of PrP 27–30 in the brains of terminally ill Syrian hamsters.


Prion Protein Multiple Reaction Monitoring Scrapie PITC Fatal Neurodegenerative Disease 
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  1. 1.
    Prusiner, S. B. Novel Proteinaceous Infectious Particles Cause Scrapie. Science 1982, 216(4542), 136–144.CrossRefGoogle Scholar
  2. 2.
    Prusiner, S. B. Prions. Proc. Natl. Acad. Sci. U.SA. 1998, 95(23), 13363–13383.CrossRefGoogle Scholar
  3. 3.
    Soto, C. Diagnosing Prion Diseases: Needs, Challenges and Hopes. Nat. Rev. Microbiol. 2004, 2(10), 809–819.CrossRefGoogle Scholar
  4. 4.
    Aguzzi, A. M. Polymenidou, M. Mammalian Prion Biology: One Century of Evolving Concepts. Cell. 2004, 116(2), 313–327.CrossRefGoogle Scholar
  5. 5.
    Prusiner, S. B. Molecular Biology of Prion Diseases. Science 1991, 252(5012), 1515–1522.CrossRefGoogle Scholar
  6. 6.
    Stahl, N. Baldwin, M. A. Teplow, D. B. Hood, L. Gibson, B. W. Burlingame, A. L. Prusiner, S. B. Structural Studies of the Scrapie Prion Protein Using Mass Spectrometry and Amino Acid Sequencing. Biochemistry 1993, 32(8), 1991–2002.CrossRefGoogle Scholar
  7. 7.
    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. Biochemistry 1992, 31(21), 5043–5053.CrossRefGoogle Scholar
  8. 8.
    Stahl, N. Baldwin, M. A. Prusiner, S. B. Electrospray Mass Spectrometry of the Glycosylinositol Phospholipid of the Scrapie Prion Protein. Cell. Biol. Int. Rep. 1991, 15(9), 853–862.CrossRefGoogle Scholar
  9. 9.
    Baldwin, M. A. Stahl, N. Reinders, L. G. Gibson, B. W. Prusiner, S. B. Burlingame, A. L. Permethylation and Tandem Mass Spectrometry of Oligosaccharides Having Free Hexosamine: Analysis of the Glycoinositol Phospholipid Anchor Glycan from the Scrapie Prion Protein. Anal. Biochem. 1990, 191(1), 174–182.CrossRefGoogle Scholar
  10. 10.
    Stahl, N. Baldwin, M. A. Burlingame, A. L. Prusiner, S. B. Identification of Glycoinositol Phospholipid Linked and Truncated Forms of the Scrapie Prion Protein. Biochemistry 1990, 29(38), 8879–8884.CrossRefGoogle Scholar
  11. 11.
    Onisko, B. Fernandez, E. G. Freire, M. L. Schwarz, A. Baier, M. Camina, F. Garcia, J. R. Rodriguez-Segade Villamarin, S. Requena, J. R. Probing PrPSc Structure Using Chemical Cross-Linking and Mass Spectrometry: Evidence of the Proximity of Gly90 Amino Termini in the PrP 27–30 Aggregate. Biochemistry 2005, 44(30), 10100–10109.CrossRefGoogle Scholar
  12. 12.
    Hunter, N. Foster, J. Chong, A. McCutcheon, S. Parnham, D. Eaton, S. MacKenzie, C. Houston, F. Transmission of Prion Diseases by Blood Transfusion. J. Gen. Virol. 2002(Pt 11, 83, 2897–2905.Google Scholar
  13. 13.
    Prusiner, S. B. McKinley, M. P. Bowman, K. A. Bolton, D. C. Bendheim, P. E. Groth, D. F. Glenner, G. G. Scrapie Prions Aggregate to Form Amyloid-Like Birefringent rods. Cell. 1983, 35(2 Pt 1), 349–358.CrossRefGoogle Scholar
  14. 14.
    Brown, P. L. Cervenakova, L. Diringer, H. Blood Infectivity and the Prospects for a Diagnostic Screening Test in Creutzfeldt-Jakob Disease. J. Lab. Clin. Med. 2001, 137(1), 5–13.CrossRefGoogle Scholar
  15. 15.
    Prusiner, S. B. Cochran, S. P. Groth, D. F. Downey, D. E. Bowman, K. A. Martinez, H. M. Measurement of the Scrapie Agent Using an Incubation Time Interval Assay. Ann. Neurol. 1982, 11(4), 353–358.CrossRefGoogle Scholar
  16. 16.
    Wadsworth, J. D. Joiner, S. Hill, A. F. Campbell, T. A. Desbruslais, M. Luthert, P. J. Collinge, J. Tissue Distribution of Protease Resistant Prion Protein in Variant Creutzfeldt-Jakob Disease Using a Highly Sensitive Immunoblotting Assay. Lancet. 2001, 358(9277)), 171–180.CrossRefGoogle Scholar
  17. 17.
    Zanusso, G. Righetti, P. G. Ferrari, S. Terrin, L. Farinazzo, A. Cardone, F. Pocchiari, M. Rizzuto, N. Monaco, S. Two-Dimensional Mapping of Three Phenotype-Associated Isoforms of the Prion Protein in Sporadic Creutzfeldt-Jakob Disease. Electrophoresis 2002, 23(2), 347–355.CrossRefGoogle Scholar
  18. 18.
    Lee, D. C. Stenland, C. J. Hartwell, R. C. Ford, E. K. Cai, K. Miller, J. L. Gilligan, K. J. Rubenstein, R. Fournel, M. Petteway, S. R., Jr. Monitoring Plasma Processing Steps With a Sensitive Western Blot Assay for the Detection of the Prion Protein. J. Virol. Methods. 2000, 84(1), 77–89.CrossRefGoogle Scholar
  19. 19.
    Deslys, J. P. Comoy, E. Hawkins, S. Simon, S. Schimmel, H. Wells, G. Grassi, J. Moynagh, J. Screening Slaughtered Cattle for BSE. Nature 2001 409(6819), 476–478.CrossRefGoogle Scholar
  20. 20.
    Biffiger, K. Zwald, D. Kaufmann, L. Briner, A. Nayki, I. Purro, M. Bottcher, S. Struckmeyer, T. Schaller, O. Meyer, R. Fatzer, R. Zurbriggen, A. Stack, M. Moser, M. Oesch, B. Kubler, E. Validation of a Luminescence Immunoassay for the Detection of PrP(Sc) in Brain Homogenate. J. Virol. Methods. 2002, 101(1/2), 79–84.CrossRefGoogle Scholar
  21. 21.
    Grassi, J. Comoy, E. Simon, S. Creminon, C. Frobert, Y. Trapmann, S. Schimmel, H. Hawkins, S. A. Moynagh, J. Deslys, J. P. Wells, G. A. Rapid Test for the Preclinical Post Mortem Diagnosis of BSE in Central Nervous System Tissue. Vet. Rec. 2001, 149(19), 577–582.CrossRefGoogle Scholar
  22. 22.
    Safar, J. Wille, H. Itri, V. Groth, D. Serban, H. Torchia, M. Cohen, F. E. Prusiner, S. B. Eight Prion Strains Have PrP(Sc) Molecules with Different Conformations. Nat. Med. 1998, 4(10), 1157–1165.CrossRefGoogle Scholar
  23. 23.
    Klohn, P. C. Stoltze, L. Flechsig, E. Enari, M. Weissmann, C. A Quantitative, Highly Sensitive Cell-Based Infectivity Assay for Mouse Scrapie Prions. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(20), 11666–11671.CrossRefGoogle Scholar
  24. 24.
    Castilla, J. Saa, P. Soto, C. Detection of Prions in Blood. Nat. Med. 2005, 11(9), 982–985.Google Scholar
  25. 25.
    Supattapone, S. Prion Protein Conversion in Vitro. J. Mol. Med. 2004, 82(6), 348–356.CrossRefGoogle Scholar
  26. 26.
    Lu, Y. Bottari, P. Turecek, F. Aebersold, R. Gelb, M. H. Absolute Quantification of Specific Proteins in Complex Mixtures Using Visible Isotope-Coded Affinity Tags. Anal. Chem. 2004, 76(14), 4104–4111.CrossRefGoogle Scholar
  27. 27.
    Gerber, S. A. Rush, J. Stemman, O. Kirschner, M. W. Gygi, S. P. Absolute Quantification of Proteins and Phosphoproteins from Cell Lysates by Tandem MS. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(12), 6940–6945.CrossRefGoogle Scholar
  28. 28.
    Sidhu, K. S. Sangvanich, P. Brancia, F. L. Sullivan, A. G. Gaskell, S. J. Wolkenhaue, O. Oliver, S. G. Hubbard, S. J. Bioinformatic Assessment of Mass Spectrometric Chemical Derivatization Techniques for Proteome Database Searching. Proteomics. 2001, 1(11), 1368–1377.CrossRefGoogle Scholar
  29. 29.
    van der Rest, G. He, F. Emmett, M. R. Marshall, A. G. Gaskell, S. J. Gas-Phase Cleavage of PTC-Derivatized Electrosprayed Tryptic Peptides in an FT-ICR Trapped-Ion Cell: Mass-Based Protein Identification Without Liquid Chromatographic Separation. J. Am. Soc. Mass Spectrom. 2001, 12(3), 288–295.CrossRefGoogle Scholar
  30. 30.
    Diringer, H. Beekes, M. Ozel, M. Simon, D. Queck, I. Cardone, F. Pocchiari, M. Ironside, J. W. Highly Infectious Purified Preparations of Disease-Specific Amyloid of Transmissible Spongiform Encephalopathies Are Not Devoid of Nucleic Acids of Viral Size. Intervirology 1997, 40(4), 238–246.CrossRefGoogle Scholar
  31. 31.
    Laemmli, U. K. Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 1970, 227(5259), 680–685.CrossRefGoogle Scholar
  32. 32.
    Tholey, A. Wittmann, C. Kang, M. J. Bungert, D. Hollemeyer, K. Heinzle, E. Derivatization of Small Biomolecules for Optimized Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom. 2002, 37(9), 963–973.CrossRefGoogle Scholar
  33. 33.
    Ducret, A. Bures, E. J. Aebersold, R. High-Sensitivity Detection of 4-(3-Pyridinylmethylaminocarboxypropyl) Phenylthiohydantoins by Capillary Liquid Chromatography-Microelectrospray Ion Trap Mass Spectrometry. J. Protein Chem. 1997, 16(5), 323–328.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Bruce Onisko
    • 1
  • Irina Dynin
    • 1
  • Jesús R. Requena
    • 2
  • Christopher J. Silva
    • 1
  • Melissa Erickson
    • 1
  • John Mark Carter
    • 1
  2. 2.Prion Research Unit, Department of Medicine, School of MedicineUniversity of SantiagoSantiagoSpain

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