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

Abstract

Scrapie is a prion (PrP) 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). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [MLGSXMSRPL(X = A or V), YXENMY(X,= H or R), and YRPVDXY(X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPor PrP. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPisolated from sheep heterozygous for their PrPproteins. 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. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrPand samples containing only the PK-resistant PrP. These results show that heterozygous animals contain PrPthat is composed of significant amounts of both PrP polymorphisms.