A mass spectrometry-based method of quantifying the contribution of the lysine polymorphism at position 171 in sheep PrP

Authors: Silva, Christopher - Chris; Cassmann, Eric; Greenlee, Justin; Erickson-Beltran, Melissa; REQUENA, JESUS - University Of Santiago De Compostela

Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2022
Publication Date: 1/9/2023
Citation: Silva, C.J., Cassmann, E.D., Greenlee, J.J., Erickson-Beltran, M.L., Requena, J.R. 2023. A mass spectrometry-based method of quantifying the contribution of the lysine polymorphism at position 171 in sheep PrP. Journal of American Society for Mass Spectrometry. 34(2):245-254. https://doi.org/10.1021/jasms.2c00277.
DOI: https://doi.org/10.1021/jasms.2c00277

Interpretive Summary: Scrapie is a prion disease that has an adverse economic impact on the US sheep industry. Scrapie is caused by a natively expressed protein (PrPC) that has been reshaped into a prion. The ability of PrPC to adopt the prion shape and cause disease progression is strongly dependent upon the amino acid at position 171 of PrPC. Sheep expressing lysine (K) at position 171 (K171) are highly resistant to scrapie infection when experimentally fed scrapie. K171 sheep are, however, susceptible to scrapie when experimentally inoculated with it, but the resulting disease progresses much slower than would be expected. Heterozygous sheep, expressing glutamine (Q) at position 171 (Q171) in one copy of the protein and K171 in the other, are partially resistant to scrapie when experimentally fed scrapie. It is not known what portion of a scrapie-infected heterozygous (Q171/K171) sheep’s prions are K171. We used stable isotope (15N)-labeled and other recombinant prion proteins (rPrP) to develop a mass spectrometry-based method to detect the amount of K171. The method was verified using rPrP. In addition, we developed methods to isolate scrapie prions that minimize the presence of background molecules that interfere with this analysis. In the future we plan to use this method to measure the amount of lysine in scrapie from heterozygous (Q171/K171) sheep experimentally infected with scrapie.

Technical Abstract: In sheep, the transmissibility and progression of scrapie, a sheep prion (PrPSc) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrPC). Sheep expressing PrPC with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrPSc. A tryptic peptide, YPNQVYYRPVDK, derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring (MRM)-based analysis. This method, using 15N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie infected sheep. Isolation methods were employed to minimize the contribution of those background molecules, making this approach useful for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep.