Location: Produce Safety and Microbiology ResearchTitle: A general mass spectrometry-based method of quantitating the oxidation of the methionine side chains in classical and atypical scrapie PrPSc
|Silva, Christopher - Chris|
|MARTIN-BURRIEL, INMACULADA - University Of Zaragoza|
|BADIOLA, JUAN - University Of Zaragoza|
|REQUENA, JESUS - University Of Santiago De Compostela|
|BOLEA, ROSA - University Of Zaragoza|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/29/2019
Publication Date: 5/18/2019
Citation: Silva, C.J., Erickson-Beltran, M.L., Martin-Burriel, I., Badiola, J.J., Requena, J.R., Bolea, R. 2019. 238: A general mass spectrometry-based method of quantitating the oxidation of the methionine side chains in classical and atypical scrapie PrPSc [abstract]. Prion. 13(Supplement):131-132.
Technical Abstract: Background/Introduction: The structure of PrPSc is uncertain, but the most compelling data suggests that it is a four-rung beta-solenoid. The rungs of the solenoid are beta-sheet secondary structures held together by hydrogen bonds. Unstructured loops connect the more rigid beta-sheet structures. The side chains of the amino acids in the beta-sheets alternately project inside the solenoid or outside of it. This may make the reactivity of adjacent amino side chains dramatically different. The side chain of methionine can be oxidized under physiological conditions to the much more polar sulfoxide. Eukaryotic cells possess enzymes that reduce oxidized methionines, so the methionines of PrPC should be largely unoxidized. Methionines present in PrPSc may be oxidized by chemical reagents or natural oxidative processes. Quantitating the extent of this oxidation can provide insight into the location of the methionines within the beta-solenoid. Materials and Methods: Plasmids containing relevant PrP sequences were prepared. 15N-labeled internal standards were prepared by overexpressing the PrP genes in E. coli grown in minimal medium with 15NH4Cl as the sole nitrogen source. A set of peptides containing the methionines was identified that are derived from the tryptic or chymotryptic digestion of sheep PrP. A multiple reaction monitoring method (MRM) was developed to detect and quantitate each peptide. Results: Calibration curves relating signal intensities of the various peptides and their absolute amounts were linear and had excellent correlation coefficients. The optimal digestion for all tryptic peptides was 20 hours. For chymotrypsin the optimal digestion was 20 hours, except for one peptide. In peptides containing two methionines, the oxidation state of each could be readily identified by a combination of chromatographic properties and the MRM method. Conclusions: This approach can be used to quantitate the relative amounts of each oxidized methionine in sheep scrapie. By comparing the results obtained from classical and atypical scrapie, we gained insight into the structural differences between the two isoforms.