Location: Produce Safety and Microbiology ResearchTitle: Using mass spectrometry to quantify prion polymorphisms and phenotypes in heterozygous sheep and deer
|Silva, Christopher - Chris
|DUQUE-VELÁSQUEZ, CAMILO - University Of Alberta
|AIKEN, JUDD - University Of Alberta
|MCKENZIE, DEBBIE - University Of Alberta
|MARTÍN-BURRIEL, INMACULADA - University Of Zaragoza
|BADIOLA JUAN, JOSÉ - University Of Zaragoza
|REQUENA, JESÚS - University Of Santiago De Compostela
|MARÍN, BELÉN - University Of Zaragoza
|BOLEA, ROSA - University Of Zaragoza
Submitted to: American Chemical Society
Publication Type: Abstract Only
Publication Acceptance Date: 6/9/2020
Publication Date: 8/17/2020
Citation: C.J. Silva, M.L. Erickson-Beltran, C. Duque-Velásquez, J.M. Aiken, D. Mckenzie, I. Martín-Burriel, J. Badiola, J.R. Requena, B. Marín, R. Bolea. 2020. AGFD 138: Using mass spectrometry to quantify prion polymorphisms and phenotypes in heterozygous sheep and deer. Abstracts of Papers of the American Chemical Society. ACS Fall 2020 Virtual Meeting & Exposition: 138-AGFD.
Technical Abstract: Prions (PrPSc) are molecular pathogens that replicate by inducing a natively expressed cellular prion protein (PrPC) to adopt the prion conformation. The efficiency of this conversion is highly dependent on the sequence of PrPC. Homozygous sheep expressing arginine (R) at position 171 are resistant to classical scrapie. Such sheep, however, are highly susceptible to a novel sporadic scrapie strain referred to as Nor98, or atypical scrapie. Sheep and cervids (deer, elk, moose, etc.) each express at least 20 different PrPC polymorphisms. Unlike sheep, none of the cervid polymorphisms completely protect against chronic wasting disease (CWD) infection. PrPC contains post-translational modifications, variable asparagine glycosylation and a glycosylphosphatidylinositol (GPI)-anchor, which preclude a simple whole-protein-based analysis. Trypsin, chymotrypsin, or a sequential digestion of both enzymes was, therefore, used to generate a set of peptides suitable for a multiple reaction monitoring (MRM)-based analysis that spanned the known sheep and cervid PrPC polymorphic sites. Calibration curves relating the area ratios of the MRM signals from polymorphism-containing peptides to internal standard peptides were linear with excellent correlation coefficients. This approach was used to measure the relative amounts of PrP polymorphisms in heterozygous sheep naturally infected with classical scrapie and in deer experimentally infected with CWD. The same approach was also used to measure the relative propagation of PrP polymorphisms in heterozygous sheep naturally infected atypical scrapie. It was also used to quantify the relative amount of PrP polymorphisms in CWD strains whose propagation properties are substantially altered. In this way, mass spectrometry can be used to quantitate phenotypic differences in classical and atypical scrapie and CWD prions.