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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #361313

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: A general mass spectrometry-based method of quantitating the relative amounts of PrP polymorphisms in CWD PrPSc

item Silva, Christopher - Chris
item Erickson-Beltran, Melissa
item DUQUE VELASQUEZ, CAMILO - University Of Alberta
item AIKEN, JUDD - University Of Alberta
item MCKENZIE, DEBBIE - University Of Alberta

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/29/2019
Publication Date: 5/18/2019
Citation: Silva, C.J., Erickson-Beltran, M.L., Duque Velasquez, C., Aiken, J.M., Mckenzie, D. 2019. 237: A general mass spectrometry-based method of quantitating the relative amounts of PrP polymorphisms in CWD PrPSc [abstract]. Prion. 13(supplement):131-131.

Interpretive Summary:

Technical Abstract: Background/Introduction: Chronic wasting disease (CWD) was first described in a captive cervid herd in 1967, recognized as a prion disease in 1978, and first detected in wild cervids in 1981. CWD is readily transmitted among animals and from a contaminated environment to uninfected animals. By 2019, 26 states in the United States, three Canadian provinces, South Korea, Norway, and Finland had found CWD in captive and/or wild cervid populations. Cervid PrP contains polymorphisms that influence progression of CWD and propagation of CWD strains with different transmission properties.1-3 It is important to develop a method to quantitate the relative amounts of those polymorphisms present in CWD PrPSc, so that their respective influence on CWD prion propagation can be determined. Materials and Methods: Plasmids containing relevant cervid PrP polymorphisms were prepared. The necessary 15N-labeled internal standards were produced by overexpressing the PrP genes in E. coli grown in minimal medium with 15NH4Cl as the sole nitrogen source. Peptides derived from the tryptic or chymotryptic digestion of cervid recombinant PrP and containing the relevant polymorphisms were identified. A multiple reaction monitoring method (MRM) was developed for each of those peptides. Results: Calibration curves relating the signal intensity of the various peptides and their absolute amounts as measure by protein assay were linear and had excellent correlation coefficients. In addition, samples from experimentally infected heterozygous deer (Q95S96/Q95G96 and H95G96 /Q95G96) were analyzed and the relative amounts were determined. The optimal digestion for all tryptic peptides was 20 hours. For chymotrypsin the optimal digestion time was also 20 hours, apart from a single peptide. Conclusions: This approach can be used to quantify the contribution of specific polymorphisms to PrPCWD formation, enhancing our ability to determine the relative susceptibility of various polymorphisms to CWD. It can also be used to detect the distribution of CWD prion strains. References 1) Johnson CJ, et al. PLoS ONE 2011; 6:e17450. 2) Duque Velasquez C, et al., (2015) J Virol 2015; 89:12362-73. 3) Herbst A, et al. Emerg Infect Dis 2017; 23:1598-1600.