A Mass Spectrometry-Based Method of Quantifying the Contribution of the Lysine Polymorphism at Position 171 in Sheep PrP

• Published in: Journal of the American Society for Mass Spectrometry Vol. 34; no. 2; pp. 245 - 254
• Main Authors: Silva, Christopher J., Cassmann, Eric D., Greenlee, Justin J., Erickson-Beltran, Melissa L., Requena, Jésus R.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.02.2023
• Subjects: Animals; Brain - metabolism; Endopeptidase K; Lysine - metabolism; Mass Spectrometry; Prion Proteins; Scrapie - genetics; Scrapie - metabolism; Sheep; recombinant prion protein; multiple reaction monitoring; TSE; prion; mass spectrometry; rPrP; transmissible spongiform encephalopathy; scrapie; 171 polymorphism
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1021/jasms.2c00277

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, 159R.YPNQVYYRPVDK.Y172, derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-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. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep.