A general mass spectrometry-based method of quantitating prion polymorphisms from heterozygous chronic wasting disease-infected cervids

Authors: Silva, Christopher - Chris; Erickson-Beltran, Melissa; DUQUE VELÁSQUEZ, CAMILO - University Of Alberta; AIKEN, JUDD - University Of Alberta; MCKENZIE, DEBBIE - University Of Alberta

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2019
Publication Date: 12/9/2019
Citation: Silva, C.J., Erickson-Beltran, M.L., Duque Velásquez, C., Aiken, J.M., McKenzie, D. 2019. A general mass spectrometry-based method of quantitating prion polymorphisms from heterozygous chronic wasting disease-infected cervids. Analytical Chemistry. 92(1):1276-1284. https://doi.org/10.1021/acs.analchem.9b04449.
DOI: https://doi.org/10.1021/acs.analchem.9b04449

Interpretive Summary: Chronic wasting disease (CWD) is naturally transmitted among farmed and free-ranging cervids (deer, elk, moose, etc.). It has a long asymptomatic incubation period, followed by a short symptomatic phase that inevitably ends in the death of the infected animal. Even though a CWD-infected cervid may appear to be healthy, it can still transmit CWD to uninfected animals. CWD-infected animals can contaminate their environment resulting in the infection of other animals by their interaction with that contaminated environment. As of September 2019, CWD-infected wild cervids have been found in 24 states, three Canadian provinces, Korea, Norway, Finland, and Sweden. CWD is caused by a pathological protein called a prion. Prions (PrPSc) replicate by inducing a normal cellular prion protein (PrPC) to adopt the prion shape. This prion shape-mediated conversion is strongly influenced by differences in the components that make up PrPC (amino acid polymorphisms). Cervid PrPC contains at least 18 different amino acid polymorphic sites. We developed a general method to measure the relative abundance of the polymorphisms at each of these sites. We did this by using specific enzymes (chymotrypsin, trypsin, or both) to digest PrPC into a group of 19 peptides which could be analyzed and contained all of the polymorphic sites. We used 10 of these peptides as internal standards to determine the relative amount of the polymorphism-containing peptides. Animals with only the amino acid serine at position 96 (S96) are resistant to infection with CWD from animals with only the amino acid glycine at position 96 (G96). Heterozygous white-tailed deer, producing both G96 and S96, were experimentally infected with CWD from a deer that only produced G96. To test our method, we isolated prions from those heterozygous white-tailed deer and analyzed them. We found that isolated prions contained 75% G96 and 25% S96. This indicates that heterozygous animals convert either of the PrPC polymorphisms into PrPSc, which means that heterozygous animals can help prions adapt to infect CWD-resistant animals. This approach can be used to determine the relative amounts of the polymorphisms present in other animal species and even humans.

Technical Abstract: Chronic wasting disease (CWD) is the only prion disease naturally transmitted among farmed and free-ranging animals (cervids, deer, elk, moose, etc.). These diseases are always fatal and have long asymptomatic incubation periods. By 2019, CWD-infected cervids had been detected in 26 states, three Canadian provinces, South Korea, Norway, Finland, and Sweden. Prions (PrPSc) replicate by inducing a normal cellular prion protein (PrPC) to adopt the prion conformation. This prion templated conformational conversion is influenced by PrPC polymorphisms. Cervid PrPC contains at least 21 different polymorphic sites. Using chymotryptic, tryptic, or tryptic followed by chymotryptic digestion, a set of 18 peptides was identified that span these 21 polymorphic sites. All of these peptides are suitable for an MRM-based analysis. Seven of the 18 peptides do not possess polymorphisms and can be used as internal standards to determine the relative amount of the other polymorphism-containing peptides. Calibration curves relating the area ratios of multiple reaction monitoring (MRM) signals from polymorphism-containing peptides to the appropriate internal standard peptides were linear and had excellent correlation coefficients. Samples from heterozygous (G96/S96) white-tailed deer infected with CWD from homozygous (G96/G96) deer were analyzed. The G96 polymorphism comprised 75 ± 5% of the total PrP from the G96/S96 heterozygotes. Heterozygous animals facilitate conversion of different PrPC polymorphisms into PrPSc. This approach can be used to quantitate the relative amounts of the polymorphisms present in other animal species and even humans.