Use of methionine oxidation to study prion structure

Authors: Silva, Christopher - Chris; Erickson-Beltran, Melissa

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Abstract Only
Publication Acceptance Date: 8/10/2021
Publication Date: 11/15/2021
Citation: Silva, C.J., Erickson-Beltran, M.L. 2021. Use of methionine oxidation to study prion structure[abstract]. Citation (ID) number 308479, November 5, 2021, Proceedings of the 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia, PA and online, October 31-November 4, 2021. Available: https://www.asms.org/docs/default-source/default-document-library/tues-nov-16-12pm---para. llel-2_guide.pdf?.

Interpretive Summary:

Technical Abstract: Prions (PrPSc) are infectious proteins that propagate their pathogenicity by inducing a normal cellular prion protein (PrPC) to adopt the prion conformation. Both isoforms possess identical covalent structure and differ solely in their respective conformations. PrPC possesses a comparative abundance of methionines relative to other cellular proteins. Mammalian cells express methionine sulfoxide reductases (MsrA and MsrB) to protect cellular proteins from reactive oxygen species. Prion diseases are characterized by a long incubation period where prions could be exposed to reactive oxidative species, but access by reductases can be limited due to specific prion conformations. Experimentally oxidizing prions does not eliminate their infectivity. Quantifying the extent of methionine oxidation in prions can provide information about the characteristic prion conformation. Methods Recombinant hamster PrP (rHaPrP) was prepared by overexpressing the hamster Prnp gene. rHaPrP was prepared from E. coli grown in minimal medium containing natural abundance ammonium chloride (14NH4Cl). The necessary internal standards were prepared from E. coli grown in minimal medium supplemented with 15NH4Cl. Suitable synthetic peptides were obtained from a commercial vendor and optimized for a multiple reaction monitoring (MRM)-based analysis.Peptides derived from the proteolytic digestion of either 14N-HarPrP, 15N-HarPrP or hamster prions were analyzed by MRM. Fixed amounts of commercially synthesized peptides were used with 15N-labeled internal standards to prepare calibration curves, which were used to quantify the relative amounts of methionine oxidation present in rHaPrP and hamster prion samples. Preliminary data The amino acid sequence of hamster PrPC was analyzed by Expasy to identify proteases (trypsin, chymotrypsin, trypsin+chymotrypsin) to digest hamster PrP into suitable peptides containing one or two of the nine methionines present in the protein. The in silico analysis of rHaPrP digestion revealed seven peptides (TNMK, HMAGAAAAGAVVGGLGGY, MLGSAMSR, PMMHFGNDWEDR, ENMNR, IMER, VVEQMCTTQYQK)that were suitable for an MRM-based analysis. rHaPrP was prepared in either natural abundance (14N) or stable isotope labeled (15N) form. Each was digested with either trypsin,chymotrypsin or both enzymes. The resulting digests were analyzed to verify that the desired seven peptides could be obtained. Each peptide was obtained from a commercial vendor and optimized for MRM-based analysis. The chromatography enabled baseline separation of the methionine from the methionine sulfoxide. Two peptides (MLGSAMSR and PMMHFGNDWEDR) contain two methionines each. Our analysis showed that the native peptide, each of the monoxidized peptides and the doubly oxidized peptides had a baseline separation. Optimal digest conditions were determined for both rHaPrP and hamster prions. These conditions permit the analysis of either source of prion protein. In this way we can accurately quantitate the relative extent of methionine oxidation for each methionine in the seven peptides and by extension the nine methionines in hamster PrP. By comparing the methionine oxidation in rHaPrP, which possesses the same conformation as hamster PrPC, to that in hamster prions, we can determine which methionines are more surface exposed and thereby infer conformational differences. Novel aspect Quantifying the extent of methionine oxidation and mapping more heavily oxidized methionines provides information about the prion conformation.