General Method of Quantifying the Extent of Methionine Oxidation in the Prion Protein

Abstract

The normal cellular prion protein (PrP^C) and its infectious conformer, PrP^Sc, possess a disproportionately greater amount of methionines than would be expected for a typical mammalian protein. The thioether of methionine can be readily oxidized to the corresponding sulfoxide, which means that oxidation of methionine can be used to map the surface of the conformation of PrP^C or PrP^Sc, as covalent changes are retained after denaturation. We identified a set of peptides (TNMK, MLGSAMSR, LLGSAMSR, PMIHFGNDWEDR, ENMNR, ENMYR, IMER, MMER, MIER, VVEQMCVTQYQK, and VVEQMCITQYQR) that contains every methionine in sheep, cervid, mouse, and bank vole PrP. Each is the product of a tryptic digestion and is suitable for a multiple reaction monitoring (MRM) based analysis. The peptides chromatograph well. The oxidized and unoxidized peptides containing one methionine readily separate. The unoxidized, two singly oxidized, and doubly oxidized forms of the MLGSAMSR and MMER peptides are also readily distinguishable. This approach can be used to determine the surface exposure of each methionine by measuring its oxidation after reaction with added hydrogen peroxide.

Keywords: TSE; mass spectrometry; methionine; methionine sulfoxide; multiple reaction monitoring; prion; rPrP; recombinant prion protein; transmissible spongiform encephalopathy.