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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #350295

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: Does methionine oxidation influence the progression of classical or atypical scrapie

Author
item Silva, Christopher - Chris
item Erickson-Beltran, Melissa
item MARTÍN-BURRIEL, INMACULADA - University Of Zaragoza
item BADIOLA, JUAN JOSÉ - University Of Zaragoza
item REQUENA, JESUS - University Of Santiago De Compostela
item BOLEA, ROSA - University Of Zaragoza

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/6/2018
Publication Date: 6/7/2018
Citation: Silva, C.J., Erickson-Beltran, M.L., Martín-Burriel, I., Badiola, J., Requena, J.R., Bolea, R. 2018. Does methionine oxidation influence the progression of classical or atypical scrapie. 66thASMS Conference on Mass Spectrometry and Allied Topics. Poster No. ThP 586. June 7, 2018. San Diego California.

Interpretive Summary: Scrapie is a fatal prion disease of sheep. There are two kinds of scrapie, classical which is transmitted among sheep and atypical, which is thought to arise spontaneously. Prions are pathological proteins that induce a normal cellular prion protein to adopt the distinct shape of a prion, resulting in characteristic brain infection of sheep. Portions of the normal cellular prion protein can be oxidized. It is possible that this oxidation could make the conversion of normal cellular prion protein to prion easier. We developed a method of measuring the amount of oxidation present in prions using a scientific instrument (mass spectrometer). This instrument allowed us to measure the amount of oxidation present in specific parts of the prion. We found that most of the prion was not oxidized. Some of the parts were consistently more oxidized than other parts. Some of the samples were more oxidized than others. The reasons for these differences are not clear and may reflect the way the samples were obtained.

Technical Abstract: Introduction Prions are pathological proteins that propagate by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). PrPC and PrPSc possess identical covalent structures and only differ in their conformations. The conversion of the PrPC conformation to the PrPSc one is template driven and highly dependent on the amino acid sequence of PrPC. Classical scrapie is the prototypical prion disease, known since the 18th century and transmitted amongst sheep and goats. Atypical scrapie was discovered in the late 20th century and is thought to be a spontaneous disease. Oxidation of methionine has been implicated in the formation of PrPSc. We determined the extent of methionine oxidation in PrPSc from classical and atypical scrapie samples and discuss its importance. Methods PrPSc samples were isolated from sheep naturally infected with classical and atypical scrapie. The isolated PrPSc was digested with trypsin or chymotrypsin to yield sets of characteristic tryptic or chymotryptic peptides. These peptides were detected and quantified using a Qtrap mass spectrometer. The instrument parameters were optimized for each peptide and used to develop multiple reaction monitoring methods (MRMs). Suitable 15N-labeled recombinant proteins were isolated from clones and digested with trypsin or chymotrypsin to yield the required 15N-labeled internal standards. Fixed amounts of commercially synthesized peptides were used with these added internal standards to prepare calibration curves. These curves were used to quantify the relative amounts of methionine oxidation present in scrapie-infected samples. Preliminary data In silico digestions of sheep prion protein (PrP) indicated that suitable peptides spanning the 136 (A/V), 154 (H/R), and 171 (R/Q/H/K) polymorphisms associated with classical and atypical scrapie could be obtained by digestion with chymotrypsin.Experimental digestions of recombinant PrP (rPrP) were performed and analyzed with an Orbitrap Elite mass spectrometer to confirm the in silico predictions. This analysis identified the methionine containing chymotryptic peptides M132LGSXMSRPL141 (X136=A or V) and Y153XENMY158 (X154=H or R). Synthetic peptides were obtained from a commercial vendor and then optimized for use in a multiple reaction monitoring (MRM) method employing a Qtrap mass spectrometer. 15N-labeled sheep rPrP was produced for use as an internal standard. Calibration curves relating the amount of each peptide to its 15N-labeled analog were prepared for all of the peptides. These curves were linear with excellent correlation coefficients, and allowed for the quantitation of the four peptides. Although the number of peptides that were used in this study was large, none interfered with the analysis of the others. Transitions were included to permit an accounting of individual methionine oxidation. Peptides derived from classical and atypical scrapie prions were analyzed using this MRM method. Methionine oxidation of the Y153XENMY158 (X154=H or R) peptide was found to be less than 10% for most samples. The M132LGSXMSRPL141 (X136=A or V) peptide contains two methionines, giving rise to four oxidative states: unoxidized, singly oxidized at M132, singly oxidized at M137, or doubly oxidized (M132 and M137). The unoxidized form predominated in most samples, though where oxidation was found, the M137 was found to be more heavily oxidized than M132. Oxidation levels were significantly higher for some individual scrapie samples, but this was not correlated with the type of scrapie, either atypical or classical. Novel aspect: A method was developed to rapidly analyze multiple oxidized methionines and assess their influence on prion replication.