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Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep
Christopher J Silva1, Melissa L Erickson-Beltran1, Colleen Hui1
1Produce Safety & Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service , Albany, California 94710, United States.
Analytical Chemistry
|December 13, 2016
Summary
Sheep scrapie disease is linked to prion protein (PrP) gene variations. A new method detects both PrP polymorphisms in scrapie-infected sheep, even in proteinase K-resistant forms.
Area of Science:
- Veterinary Medicine
- Molecular Biology
- Biochemistry
Background:
- Scrapie is a fatal prion disease in sheep.
- Prion protein (PrPC) gene polymorphisms at codons 136, 154, and 171 influence scrapie incubation periods.
- Understanding PrPSc composition in heterozygous sheep is crucial for disease management.
Purpose of the Study:
- To develop a method for detecting and quantifying PrP polymorphisms in sheep PrPSc.
- To analyze the PrPSc composition in heterozygous scrapie-infected sheep.
Main Methods:
- Chymotrypsin digestion of recombinant sheep PrP to identify characteristic peptides.
- Development of a multiple reaction monitoring (MRM) assay using these peptides.
- Analysis of spinal cord tissue from heterozygous scrapie-infected sheep.
Main Results:
- Identified specific peptides indicative of PrP polymorphisms at positions 136, 154, and 171.
- Achieved a detection limit of less than 50 attomoles for these peptides.
- Confirmed the presence of both PrP polymorphisms in PrPSc from heterozygous sheep, including PK-resistant forms.
Conclusions:
- The developed MRM method accurately detects PrP polymorphisms in PrPSc.
- Heterozygous sheep infected with scrapie harbor PrPSc containing significant amounts of both PrP polymorphisms.
- This finding has implications for understanding scrapie pathogenesis and diagnosis.
