|Silva, Christopher - Chris
|BADIOLA, JUAN JOSÉ - University Of Zaragoza
|REQUENA, JESÚS - Universidad De Santiago De Compostela
|BOLEA, ROSA - University Of Zaragoza
Submitted to: American Chemical Society National Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 12/22/2016
Publication Date: 4/4/2017
Citation: Silva, C.J., Erickson-Beltran, M.L., Hui, C., Badiola, J., Requena, J.R., Bolea, R. 2017. Quantifying the relative amounts of PrP polymorphisms present in prions isolated from heterozygous prion-infected animals. Abstracts of the Papers of the American Chemical Society. 253:208-BIOL.
Technical Abstract: Prions cause protein misfolding diseases, such as transmissible spongiform encephalopathy. They propagate infections by converting a normal cellular prion protein into a prion (PrPSc). PrPC and PrPSc are isosequential and differ only in their respective conformations. PrPC is monomeric and sensitive to proteinase K (PK) digestion, while PrPSc is multimeric and relatively resistant to PK. If PrPC and PrPSc have different sequences, then the incubation period of the resulting prion disease can vary depending upon the PrPC polymorphism. Sheep possess a variety of PrPC polymorphisms which influence the progression of scrapie, a sheep prion disease. The most important amino acid polymorphisms are located at positions 136, 154, and 171 of sheep PrPC. Proteolytic digestion was used to generate a set of characteristic peptides that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPC or PrPSc. The optimal fragmentation parameters for these peptides were empirically determined. These empirical parameters were used to develop a mass spectrometry-based multiple reaction monitoring (MRM) method to detect the amounts of a particular polymorphism in a sample of PrPSc. The limit of detection for these peptides was less than 50 attomoles. This MRM method was used to detect the relative amount of PrP polymorphisms present in the PrPSc from spinal cord tissue isolated from scrapie-infected sheep (Rasa Aragonesa) heterozygous for their PrPC proteins (A136R154Q171/V136R154Q171 or A136R154H171/A136R154Q171). The PrPSc from both sets of heterozygotes show the presence of both polymorphisms. If the samples were treated with proteinase K, this was also true. These results show that heterozygous animals contain PrPSc that is composed of significant amounts of both PrP polymorphisms.