Quantifying Oxidized Methionines with Mass Spectrometry to Map the Surface of Hamster Prion Strains Sc237 and 139H

Sc237 and 139H are two phenotypically different prion strains of hamster (<i>Mesocricetus auratus</i>) adapted scrapie. Each replicates by inducing the natively expressed hamster PrP^C to adopt its infectious conformation. The nine methionines in hamster PrP^C 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, TN<u><b>M</b></u>K, H<u><b>M</b></u>AGAAAAGAVVGGLGGY, <u><b>M</b></u>LGSA<u><b>M</b></u>SR, P<u><b>MM</b></u>HFGNDWEDR, EN<u><b>M</b></u>NR, I<u><b>M</b></u>ER, and VVEQ<u><b>M</b></u>CTTQYQK, 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.