posted on 2023-01-09, 17:34authored byChristopher J. Silva, Eric D. Cassmann, Justin J. Greenlee, Melissa L. Erickson-Beltran, Jésus R. Requena
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.