A General Mass Spectrometry-Based Method of Quantitating Prion Polymorphisms from Heterozygous Chronic Wasting...

Chronic wasting disease (CWD) is the only prion disease naturally transmitted among farmed and free-ranging 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 (PrP^Sc) replicate by inducing a normal cellular prion protein (PrP^C) to adopt the prion conformation. This prion templated conformational conversion is influenced by PrP^C polymorphisms. Cervid PrP^C contains at least 20 different polymorphic sites. By using chymotrypsin, trypsin, or trypsin followed by chymotrypsin to digest denatured cervid PrP, 19 peptides suitable for multiple reaction monitoring (MRM)-based analysis and spanning positions 30-51, 61-112, and 114-231 of cervid PrP were identified. Ten of these peptides span polymorphism-containing regions of cervid PrP. The other nine contain no polymorphisms, so they can be used as internal standards. Calibration curves relating the area ratios of MRM signals from polymorphism-containing peptides to appropriate internal standard peptides were linear and had excellent correlation coefficients. Samples from heterozygous (G₉₆/S₉₆) white-tailed deer orally dosed 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 PrP^C polymorphisms into PrP^Sc. This approach can be used to quantitate the relative amounts of the polymorphisms present in other animal species and even humans.