Quantifying oxidized methionines with mass spectrometry to map the surface of hamster prion strains Sc237 and 139H

Authors: Silva, Christopher; Erickson-Beltran, Melissa

Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2026
Publication Date: 2/12/2026
Citation: Silva, C.J., Erickson-Beltran, M.L. 2026. Quantifying oxidized methionines with mass spectrometry to map the surface of hamster prion strains Sc237 and 139H. Journal of American Society for Mass Spectrometry. 37(7):1527-1535. https://doi.org/10.1021/jasms.5c00355.
DOI: https://doi.org/10.1021/jasms.5c00355

Interpretive Summary: Two different hamster-adapted prion strains (Sc237 and 139H) were analyzed. These strains replicate by inducing the normal hamster protein (PrPC) to adopt the infectious shape or conformation of the Sc237 or 139H strain. The hamster prion protein contains nine copies of the amino acid methionine. The surface exposure of a methionine determines its susceptibility to oxidation by hydrogen peroxide, which is in turn determined by the prion’s conformation. Measuring the extent of each methionine’s oxidation is a way to map the surface of a prion. The Sc237 and 139H strains were oxidized by hydrogen peroxide, unfolded, and then digested with enzymes to yield seven protein fragments or peptides, which contain hamster prion protein’s nine methionines. The seven peptides were analyzed by mass spectrometry to determine the extent of each methionine’s oxidation. This analysis revealed that the extent of oxidation for some methionines differed in the two prion strains. This was a result of the difference in the shapes of the Sc237 and 139H strains. The shapes of the hamster strains were also different from that of a sheep prion. This indicates that this approach can be used to directly compare the shapes of prion strains from different animals.

Technical Abstract: Sc237 and 139H are two phenotypically different prion strains of hamster (Mesocricetus auratus) adapted scrapie. Each replicates by inducing the natively expressed hamster PrPC to adopt its infectious conformation. The nine methionines in hamster PrPC can be oxidized to the corresponding methionine sulfoxide by hydrogen peroxide. The extent of this oxidation is determined by the methionine’s conformation-dependent surface exposure. Methionine sulfoxides are unaffected by protein denaturation. Samples of 139H and Sc237 prions were untreated or digested with proteinase K (PK), isolated by ultracentrifugation, oxidized in 0 mM or 50 mM hydrogen peroxide, inactivated by denaturation, reduced/alkylated, and then digested with trypsin, trypsin/chymotrypsin, or Arg-C. Seven peptides, TNMK, HMAGAAAAGAVVGGLGGY, MLGSAMSR, PMMHFGNDWEDR, ENMNR, IMER, and VVEQMCTTQYQK, resulted from the enzymatic digestion. These peptides contain the nine methionines in hamster PrP and were analyzed using an MRM-based approach to determine the extent of each methionine’s oxidation. Differences in the extent of methionine oxidation were observed for each strain. These differences were observed in PK digested and untreated samples. Hamster and sheep PrP share six methionines and comparison of the methionine oxidation in Sc237, 139H, and sheep scrapie showed different methionine oxidation patterns. This approach is a form of conformational sequencing that can be used to compare the surfaces of prion strains from the same species and prions from different species.