Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep

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• 作者：Christopher J. SilvaMelissa L. Erickson-Beltran ; Colleen Hui ; Juan José Badiola ; Eric M. Nicholson ; Jesús R. Requena ; Rosa Bolea
• 刊名：Analytical Chemistry
• 出版年：2017
• 出版时间：January 3, 2017
• 年：2017
• 卷：89
• 期：1
• 页码：854-861
• 全文大小：419K
• ISSN：1520-6882

文摘

Scrapie is a prion (PrP^Sc) 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 (PrP^C). 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 PrP^C or PrP^Sc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrP^Sc isolated from sheep heterozygous for their PrP^C proteins. 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 PrP^Sc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrP^Sc and samples containing only the PK-resistant PrP^Sc. These results show that heterozygous animals contain PrP^Sc that is composed of significant amounts of both PrP polymorphisms.