title: Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep.
abstract: Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep's normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M132LGSXMSRPL141(X = A or V), Y153XENMY158(X,= H or R), and Y166RPVDXY172(X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPCor PrPSc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPScisolated from sheep heterozygous for their PrPCproteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrPSc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrPScand samples containing only the PK-resistant PrPSc. These results show that heterozygous animals contain PrPScthat is composed of significant amounts of both PrP polymorphisms.
authors.affiliation: Produce Safety & Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service , Albany, California 94710, United States.
authors.forename: Christopher J
authors.identifier: 0000-0003-4521-6377
authors.initials: CJ
authors.lastname: Silva
authors.affiliation: Produce Safety & Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service , Albany, California 94710, United States.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Erickson-Beltran
authors.affiliation: Produce Safety & Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service , Albany, California 94710, United States.
authors.forename: Colleen
authors.initials: C
authors.lastname: Hui
authors.affiliation: Veterinary Faculty, Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Universidad de Zaragoza , 50013, Zaragoza Spain.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Badiola
authors.affiliation: Virus and Prion Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service , Ames, Iowa 50010, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Nicholson
authors.affiliation: CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS , Santiago de Compostela, 15782 Spain.
authors.forename: Jesús R
authors.initials: JR
authors.lastname: Requena
authors.affiliation: Veterinary Faculty, Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Universidad de Zaragoza , 50013, Zaragoza Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Bolea
mesh_terms: D000818:Animals; D011110:Polymorphism, Genetic / Q000235:genetics*; D011328:Prions / Q000032:analysis / Q000235:genetics*; D012608:Scrapie / Q000235:genetics*; D012756:Sheep; D013116:Spinal Cord / Q000737:chemistry
chemical_list: D011328:Prions