title: Utility of mass spectrometry in the diagnosis of prion diseases.
abstract: We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol. Nonanalyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice 5 weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or four mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the nonobex/nonanterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.
authors.affiliation: Western Regional Research Center, United States Department of Agriculture, Albany, California 94710, United States. christopher.silva@ars.usda.gov
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Silva
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Onisko
authors.forename: Irina
authors.initials: I
authors.lastname: Dynin
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Erickson
authors.forename: Jesús R
authors.initials: JR
authors.lastname: Requena
authors.forename: John Mark
authors.initials: JM
authors.lastname: Carter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D017096:Prion Diseases / Q000175:diagnosis*; D012015:Reference Standards; D012756:Sheep