Unraveling the details of prion (con)formation(s): Recent advances by mass spectrometry

Current Opinion in Drug Discovery and Development

Volumn 11, Issue 5, 2008, Pages 697-707

  Principe, S ^a   Maras, B ^b   Schinina M E ^b   Pocchiari, M ^a   Cardone, F ^a  

^a ISTITUTO SUPERIORE DI SANITÀ   (Italy)

^b SAPIENZA UNIVERSITY OF ROME   (Italy)

Author keywords

Diagnosis; Mass spectrometry; Molecular pathogenesis; Neurodegeneration; Prion protein; Transmissible spongiform encephalopathy

Indexed keywords

PRION PROTEIN; TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY ASSOCIATED PRION PROTEIN; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ANALYTIC METHOD; DISULFIDE BOND; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; HUMAN; IMMUNOHISTOCHEMISTRY; LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY; MATRIX ASSISTED LASER DESORPTION IONIZATION TIME OF FLIGHT MASS SPECTROMETRY; NERVE DEGENERATION; NONHUMAN; PATHOGENESIS; PHENOTYPE; PRION DISEASE; PROTEIN CONFORMATION; PROTEIN CROSS LINKING; PROTEOMICS; REVIEW; STRUCTURE ANALYSIS; TANDEM MASS SPECTROMETRY; WESTERN BLOTTING;

ANIMALS; GENOTYPE; HUMANS; MASS SPECTROMETRY; MUTATION; PHENOTYPE; POLYMORPHISM, GENETIC; PRION DISEASES; PRIONS; PROTEIN CONFORMATION; PROTEOMICS; PRPC PROTEINS; PRPSC PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 50249129328     PISSN: 13676733     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Review

References (91)

1. Pocchiari M: Prions and related neurological diseases. Mol Aspects Med (1994) 15(3):195-291.
2. Prusiner SB: Molecular biology of prion diseases. Science (1991) 252(5012):1515-1522.
3. Prusiner SB: Prions. Proc Natl Acad Sci USA (1998) 95(23):13363-13383.
4. Turk E, Teplow DB, Hood LE, Prusiner SB: Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem (1988) 176(1):21-30.
5. Bolton DC, Meyer RK, Prusiner SB: Scrapie PrP 27-30 is a sialoglycoprotein. J Virol (1985) 53(2):596-606.
6. Endo T, Groth D, Prusiner SB, Kobata A: Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry (1989) 28(21):8380-8388.
7. Baldwin MA, Stahl N, Reinders LG, Gibson BW, Prusiner SB, Burlingame AL: 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.
8. Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, Fraser PE, Kruck T, von Bohlen A, Schulz-Schaeffer W, Giese A et al: The cellular prion protein binds copper in vivo. Nature (1997) 390(6661):684-687.
9. Whittal RM, Ball HL, Cohen FE, Burlingame AL, Prusiner SB, Baldwin MA: Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Sci (2000) 9(2):332-343.
10. Kramer ML, Kratzin HD, Schmidt B, Römer A, Windl O, Liemann S, Hornemann S, Kretzschmar H: Prion protein binds copper within the physiological concentration range. J Biol Chem (2001) 276(20):16711-16719
11. Pushie MJ, Ross AR, Vogel HJ: Mass spectrometric determination of the coordination geometry of potential copper(II) surrogates for the mammalian prion protein octarepeat region. Anal Chem (2007) 79(15):5659-5667.
12. Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB: Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry (1993) 32(8):1991-2002.
13. Stimson E, Hope J, Chong A, Burlingame AL: Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions. Biochemistry (1999) 38(15):4885- 4895.
14. Rudd PM, Endo T, Colominas C, Groth D, Wheeler SF, Harvey DJ, Wormald MR, Serban H, Prusiner SB, Kobata A, Dwek RA: Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc Natl Acad Sci USA (1999) 96(23):13044-13049.
15. Baldwin MA: Analysis of glycosylphosphatidylinositol protein anchors: The prion protein. Methods Enzymol (2005) 405:172-187.
16. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS: Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry (1991) 30(31):7672-7680.
17. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, Prusiner SB: Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA (1993) 90(23):10962-10966.
18. Safar J, Roller PP, Gajdusek DC, Gibbs CJ Jr: Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J Biol Chem (1993) 268(27):20276-20284.
19. Ingrosso L, Pocchiari M, Cardone F: Bioanalytical diagnostic test for measuring prions. In: Immunoassay and Other Bioanalytical Techniques. Van Emon JM (Ed), Taylor and Francis, Boca Raton, FL, USA (2007):309-336.
20. Ladogana A, Puopolo M, Croes EA, Budka H, Jarius C, Collins S, Klug GM, Sutcliffe T, Giulivi A, Alperovitch A, Delasnerie-Laupretre N et al: Mortality from Creutzfeldt-Jakob disease and related disorders in Europe, Australia, and Canada. Neurology (2005) 64(9):1586-1591.
21. Salvatore M, Genuardi M, Petraroli R, Masullo C, D'Alessandro M, Pocchiari M: Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease. Hum Genet (1994) 94(4):375-379.
22. Mead S: Prion disease genetics. Eur J Hum Genet (2006) 14(3):273-281.
23. Hainfellner JA, Brantner-Inthaler S, Cervenáková L, Brown P, Kitamoto T, Tateishi J, Diringer H, Liberski PP, Regele H, Feucht M, Mayr N et al: The original Gerstmann-Sträussler-Scheinker family of Austria: Divergent clinicopathological phenotypes but constant PrP genotype. Brain Pathol (1995) 5(3):201-211.
24. Barbanti P, Fabbrini G, Salvatore M, Petraroli R, Cardone F, Maras B, Equestre M, Macchi G, Lenzi GL, Pocchiari M: Polymorphism at codon 129 or codon 219 of PRNP and clinical heterogeneity in a previously unreported family with Gerstmann-Sträussler-Scheinker disease (PrP-P102L mutation). Neurology (1996) 47(3):734-741.
25. Zarranz JJ, Digon A, Atarés B, Rodríguez-Martínez AB, Arce A, Carrera N, Fernández-Manchola I, Fernández- Martínez M, Fernández-Maiztegui C, Forcadas I, Galdos L et al: Phenotypic variability in familial prion diseases due to the D178N mutation. J Neurol Neurosurg Psychiatry (2005) 76(11):1491- 1496.
26. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG: Sporadic and familial CJD: Classification and characterisation. Br Med Bull (2003) 66:213-239.
27. Goldfarb LG, Petersen RB, Tabaton M, Brown P, LeBlanc AC, Montagna P, Cortelli P, Julien J, Vital C, Pendelbury WW, Haltia M et al: Fatal familial insomnia and familial Creutzfeldt-Jakob disease: Disease phenotype determined by a DNA polymorphism. Science (1992) 258(5083): 806-808.
28. Brown P, Brandel JP, Preece M, Sato T: Iatrogenic Creutzfeldt-Jakob disease: The waning of an era. Neurology (2006) 67(3):389-393.
29. Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will RG: Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet (2004) 363(9407):417-421.
30. European Centre for Disease Prevention and Control (ECDC): New case of transfusion-associated vCJD in the United Kingdom. Eurosurveillance (2006) 11(6):2895. www.eurosurveillance.org/ew/2006/060209.asp#2
31. European Centre for Disease Prevention and Control (ECDC): Fourth case of transfusion-associated v CJD infection in the United Kingdom. Eurosurveillance (2007) 12(3):3117. www.eurosurveillance.org/ ew/2007/070118.asp#4
32. Hilton DA: Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease. J Pathol (2006) 208(2):134-141.
33. Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW: Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet (2004) 364(9433):527-529.
34. Ironside JW, Bishop MT, Connolly K, Hegazy D, Lowrie S, Le Grice M, Ritchie DL, McCardle LM, Hilton D: Variant Creutzfeldt-Jakob disease: Prion protein genotype analysis of positive appendix tissue samples from a retrospective prevalence study. BMJ (2006) 332(7551):1186- 1188.
35. Brandel JP, Preece M, Brown P, Croes E, Laplanche JL, Agid Y, Will R, Alpérovitch A: Distribution of codon 129 genotype in human growth hormone-treated CJD patients in France and the UK. Lancet (2003) 362(9378):128-130.
36. Hunter N: Scrapie: Uncertainties, biology and molecular approaches. Biochim Biophys Acta (2007) 1772(6):619-628.
37. Prusiner SB: Inherited prion diseases. Proc Natl Acad Sci USA (1994) 91(11):4611-4614.
38. Gajdusek DC: Nucleation of amyloidogenesis in infectious and noninfectious amyloidoses of brain. Ann NY Acad Sci (1994) 724:173-190.
39. Brunori M, Talbot B: A mechanism for prion replication. Nature (1985) 314(6013):676.
40. Swietnicki W, Morillas M, Chen SG, Gambetti P, Surewicz WK: Aggregation and fibrillization of the recombinant human prion protein huPrP90-231. Biochemistry (2000) 39(2):424-431.
41. Kuwata K, Li H, Yamada H, Legname G, Prusiner SB, Akasaka K, James TL: Locally disordered conformer of the hamster prion protein: A crucial intermediate to PrPSc? Biochemistry (2002) 41(41):12277-12283.
42. Jarrett JT, Lansbury PT Jr: Seeding "one-dimensional crystallization" of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? Cell (1993) 73(6):1055-1058.
43. Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M, Cohen FE, DeArmond SJ, Prusiner SB: Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell (1995) 83(1):79-90.
44. Deleault NR, Harris BT, Rees JR, Supattapone S: Formation of native prions from minimal components in vitro. Proc Natl Acad Sci USA (2007) 104(23):9741-9746.
45. Schmitt-Ulms G, Legname G, Baldwin MA, Ball HL, Bradon N, Bosque PJ, Crossin KL, Edelman GM, DeArmond SJ, Cohen FE, Prusiner SB: Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein. J Mol Biol (2001) 314(5):1209-1225.
46. Satoh J, Onoue H, Arima K, Yamamura T: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein. J Neuropathol Exp Neurol (2005) 64(10):858-868.
47. Petrakis S, Sklaviadis T: Identification of proteins with high affinity for refolded and native PrPC. Proteomics (2006) 6(24):6476-6484.
48. Zomosa-Signoret V, Arnaud JD, Fontes P, Alvarez-Martinez MT, Liautard JP: Physiological role of the cellular prion protein. Vet Res (2008) 39(4):9.
49. Ghetti B, Piccardo P, Spillantini MG, Ichimiya Y, Porro M, Perini F, Kitamoto T, Tateishi J, Seiler C, Frangione B, Bugiani O et al: Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: The phenotype of the stop codon 145 mutation in PRNP. Proc Natl Acad Sci USA (1996) 93(2):744-748.
50. Gabizon R, Telling G, Meiner Z, Halimi M, Kahana I, Prusiner SB: Insoluble wild-type and protease-resistant mutant prion protein in brains of patients with inherited prion disease. Nat Med (1996) 2(1):59-64.
51. Chen SG, Parchi P, Brown P, Capellari S, Zou W, Cochran EJ, Vnencak-Jones CL, Julien J, Vital C, Mikol J, Lugaresi E et al: Allelic origin of the abnormal prion protein isoform in familial prion diseases. Nat Med (1997) 3(9):1009-1015.
52. Tagliavini F, Lievens PM, Tranchant C, Warter JM, Mohr M, Giaccone G, Perini F, Rossi G, Salmona M, Piccardo P, Ghetti B et al: A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler- Scheinker disease A117V. J Biol Chem (2001) 276(8):6009-6015.
53. Silvestrini MC, Cardone F, Maras B, Pucci P, Barra D, Brunori M, Pocchiari M: Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients. Nat Med (1997) 3(5):521-525.
54. Kitamoto T, Yamaguchi K, Doh-ura K, Tateishi J: A prion protein missense variant is integrated in kuru plaque cores in patients with Gerstmann-Sträussler syndrome. Neurology (1991) 41 (2 Pt 1):306-310.
55. Tagliavini F, Prelli F, Porro M, Rossi G, Giaccone G, Farlow MR, Dlouhy SR, Ghetti B, Bugiani O, Frangione B: Amyloid fibrils in Gerstmann- Sträussler-Scheinker disease (Indiana and Swedish kindreds) express only PrP peptides encoded by the mutant allele. Cell (1994) 79(4):695-703.
56. Parchi P, Chen SG, Brown P, Zou W, Capellari S, Budka H, Hainfellner J, Reyes PF, Golden GT, Hauw JJ, Gajdusek DC et al: Different patterns of truncated prion protein fragments correlate with distinct phenotypes in P102L Gerstmann-Sträussler-Scheinker disease. Proc Natl Acad Sci USA (1998) 95(14):8322-8327.
57. Capellari S, Cardone F, Notari S, Schininà ME, Maras B, Sità D, Baruzzi A, Pocchiari M, Parchi P: Creutzfeldt-Jakob disease associated with the R208H mutation in the prion protein gene. Neurology (2005) 64(5):905-907.
58. Schininà ME, Maras B, Cardone F, Mancone C, Principe S, Di Bari MA, Parchi P, Pocchiari M: Prion protein allotype profiling by mass spectrometry. Pure Appl Chem (2003) 75(2-3):317-323. • Describes the development of a method based on the combined use of LC and MS to reliably determine the PrP allotype profiles in TSE-affected subjects by monitoring specific reporter peptides.
59. Cartoni C, Schininà ME, Maras B, Nonno R, Vaccari G, Di Baria MA, Conte M, Liu QG, Lu M, Cardone F, Windl O et al: Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by highperformance liquid chromatography-mass spectrometry. J Chromatogr A (2005) 1081(1):122-126.
60. Ong SE, Mann M: Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol (2005) 1(5):252-262.
61. Cartoni C, Schininà ME, Maras B, Nonno R, Vaccari G, Di Bari M, Conte M, De Pascalis A, Principe S, Cardone F, Pocchiari M et al: Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci (2007) 849(1-2):302-306. •• Demonstrates for the first time the use of a LC-MS/MS protocol to quantitatively determine the pathological PrP profile in the brains of animals inoculated with an experimentally adapted scrapie strain.
62. TSE allotypes deposited in the brain of naturally scrapie-affected heterozygous sheep to elucidate the relationship between susceptibility and the presence of specific polymorphisms in these animals.
63. Bruce ME, McConnell I, Fraser H, Dickinson AG: The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: Implications for the nature of the agent and host control of pathogenesis. J Gen Virol (1991) 72(3):595-603.
64. Bessen RA, Marsh RF: Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J Virol (1992) 66(4):2096-2101.
65. Telling GC, Parchi P, DeArmond SJ, Cortelli P, Montagna P, Gabizon R, Mastrianni J, Lugaresi E, Gambetti P, Prusiner SB: Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science (1996) 274(5295):2079- 2082.
66. Caughey B, Raymond GJ, Bessen RA: Strain-dependent differences in β-sheet conformations of abnormal prion protein. J Biol Chem (1998) 273(48):32230-32235.
67. Sc molecules with different conformations. Nat Med (1998) 4(10):1157-1165.
68. Monari L, Chen SG, Brown P, Parchi P, Petersen RB, Mikol J, Gray F, Cortelli P, Montagna P, Ghetti B, Goldfarb LG et al: Fatal familial insomnia and familial Creutzfeldt-Jakob disease: Different prion proteins determined by a DNA polymorphism. Proc Natl Acad Sci USA (1994) 91(7):2839-2842.
69. Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sima AA, Trojanowski JQ, Petersen RB et al: Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neurol (1996) 39(6):767-778.
70. Zanusso G, Righetti PG, 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.
71. Zanusso G, Farinazzo A, Prelli F, Fiorini M, Gelati M, Ferrari S, Righetti PG, Rizzuto N, Frangione B, Monaco S: Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes. J Biol Chem (2004) 279(37):38936-38942.
72. Parchi P, Zou W, Wang W, Brown P, Capellari S, Ghetti B, Kopp N, Schulz-Schaeffer WJ, Kretzschmar HA, Head MW, Ironside JW et al: Genetic influence on the structural variations of the abnormal prion protein. Proc Natl Acad Sci USA (2000) 97(18):10168-10172.
73. Sc typing by N-terminal sequencing and mass spectrometry. Arch Virol Suppl (2000) (16):209-216.
74. 90 amino termini in the PrP 27-30 aggregate. Biochemistry (2005) 44(30):10100-10109. •• The combined use of chemical cross-linking, proteolytic digestion, MALDI-TOF and ESI-MS on PrP27-30 enriched fractions extracted from the brains of Syrian hamsters experimentally infected with scrapie, provided knowledge of the conformation of pathological and aggregated PrP.
75. Young MM, Tang N, Hempel JC, Oshiro CM, Taylor EW, Kuntz ID, Gibson BW, Dollinger G: High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry. Proc Natl Acad Sci USA (2000) 97(11):5802-5806.
76. Pearson KM, Pannell LK, Fales HM: Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method. Rapid Commun Mass Spectrom (2002) 16(3):149-159.
77. Sinz A: Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes. J Mass Spectrom (2003) 38(12):1225-1237.
78. Diringer H, Beekes M, Ozel M, Simon D, Queck I, Cardone F, Pocchiari M, Ironside JW: 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.
79. TSE was investigated by a combination of specific purification procedures and the use of limited proteolysis coupled with MS analysis.
80. Neurath H: Limited proteolysis, protein folding and physiological regulation. In: Protein Folding. Jaenicke R (Ed), Elsevier, Amsterdam, The Netherlands (1980):501-523.
81. Hubbard SJ: The structural aspects of limited proteolysis of native proteins. Biochim Biophys Acta (1998) 1382(2):191-206.
82. Kheterpal I, Williams A, Murphy C, Bledsoe B, Wetzel R: Structural features of the Aβ amyloid fibril elucidated by limited proteolysis. Biochemistry (2001) 40(39):11757-11767.
83. Monti M, Amoresano A, Giorgetti S, Bellotti V, Pucci P: Limited proteolysis in the investigation of β2-microglobulin amyloidogenic and fibrillar states. Biochim Biophys Acta (2005) 1753(1):44-50.
84. Govaerts C, Wille H, Prusiner SB, Cohen FE: Evidence for assembly of prions with left-handed β-helices into trimers. Proc Natl Acad Sci USA (2004) 101(22):8342-8347.
85. Daude N, Marella M, Chabry J: Specific inhibition of pathological prion protein accumulation by small interfering RNAs. J Cell Sci (2003) 116(13):2775-2779.
86. Tilly G, Chapuis J, Vilette D, Laude H, Vilotte JL: Efficient and specific down-regulation of prion protein expression by RNAi. Biochem Biophys Res Commun (2003) 305(3):548-551.
87. Ingrosso L, Ladogana A, Pocchiari M: Congo red prolongs the incubation period in scrapie-infected hamsters. J Virol (1995) 69(1):506-508.
88. Pocchiari M, Ladogana A, Graziano S, Puopolo M: Creutzfeldt-Jakob disease: Hopes for therapy. Eur J Neurol (2008) 15(5):435-436.
89. Sanchez JC, Guillaume E, Lescuyer P, Allard L, Carrette O, Scherl A, Burgess J, Corthals GL, Burkhard PR, Hochstrasser DF: Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease. Proteomics (2004) 4(8):2229-2233. • This preliminary study identified potential biomarkers in the cerebrospinal fluid of CJD-affected patients using SELDI-TOF MS and LC-MS/MS technologies.
90. Onisko B, Dynin I, Requena JR, Silva CJ, Erickson M, Carter JM: Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS. J Am Soc Mass Spectrom (2007) 18(6):1070-1079. •• A reliable and sensitive quantitative LC-MS/MS protocol was developed to detect small amounts of PrP27-30 extracted from the brains of Syrian hamsters experimentally infected with the 263K strain.
91. Onisko BC, Silva CJ, Dynin I, Erickson M, Vensel WH, Hnasko R, Requena JR, Carter JM: Sensitive, preclinical detection of prions in brain by nanospray liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom (2007) 21(24):4023-4026. •• The PrP27-30 protein was detected in the brains of 263K-experimentally infected Syrian hamsters at an early preclinical stage (ie, 24 h after inoculation) by applying the quantitative LC-MS/MS protocol described in reference [90••].