Comparing the Extent of Methionine Oxidation in the Prion and Native Conformations of PrP.

Scrapie is a prion disease of sheep and goats. Prions (PrP) replicate by inducing a natively expressed protein (PrP) to refold into the prion conformation. PrP and PrP contain a disproportionately large number of methionines. Surface exposed methionines are more prone to chemical oxidation. Chemical oxidation is a means of measuring the surface exposure of the methionines in a prion, as these covalent changes are retained after an oxidized prion is denatured prior to analysis. Scrapie prions and recombinant sheep prion protein were oxidized in 0, 10, 20, or 50 mM solutions of hydrogen peroxide. The samples were digested with trypsin or trypsin followed by chymotrypsin to yield a set of peptides (TNMK, MLGSAMSR, ENMYR, IMER, VVEQMCITQYQR) containing the methionines present in sheep PrP. The mass spectrometry based multiple reaction monitoring (MRM) method was used to analyze these peptides. Analysis of the rPrP samples showed that surface exposed methionines (132, 137, and 157) were more oxidized than those less surface exposed (209 and 216). The extent of methionine oxidation in sheep scrapie PrP is 216 > 137 > 132 > 157 > 209 > 112. These results demonstrate that this approach can be used to map the surface exposure of the methionines in order to distinguish among PrP conformations and effect a kind of conformational sequence.