Abstract
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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Prusiner, S. B. Novel Proteinaceous Infectious Particles Cause Scrapie. Science 1982, 216(4542), 136–144.
Prusiner, S. B. Prions. Proc. Natl. Acad. Sci. U.SA. 1998, 95(23), 13363–13383.
Soto, C. Diagnosing Prion Diseases: Needs, Challenges and Hopes. Nat. Rev. Microbiol. 2004, 2(10), 809–819.
Aguzzi, A. M. Polymenidou, M. Mammalian Prion Biology: One Century of Evolving Concepts. Cell. 2004, 116(2), 313–327.
Prusiner, S. B. Molecular Biology of Prion Diseases. Science 1991, 252(5012), 1515–1522.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Castilla, J. Saa, P. Soto, C. Detection of Prions in Blood. Nat. Med. 2005, 11(9), 982–985.
Supattapone, S. Prion Protein Conversion in Vitro. J. Mol. Med. 2004, 82(6), 348–356.
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.
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.
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.
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.
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.
Laemmli, U. K. Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 1970, 227(5259), 680–685.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online April 19, 2007
Rights and permissions
About this article
Cite this article
Onisko, B., Dynin, I., Requena, J.R. et al. Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS. J Am Soc Mass Spectrom 18, 1070–1079 (2007). https://doi.org/10.1016/j.jasms.2007.03.009
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2007.03.009