General method of quantifying the extent of methionine oxidation in the prion protein

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

Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2022
Publication Date: 1/6/2023
Citation: Silva, C.J., Erickson-Beltran, M.L. 2023. General method of quantifying the extent of methionine oxidation in the prion protein. Journal of American Society for Mass Spectrometry. 34(2):255-263. https://doi.org/10.1021/jasms.2c00280.
DOI: https://doi.org/10.1021/jasms.2c00280

Interpretive Summary: The normal cellular prion protein (PrPC) is natively expressed and non-infectious. A prion (PrPSc) is an infectious protein that causes disease. PrPC and PrPSc differ only in their shapes. Determining the shape of PrPSc is important to understanding why it’s infectious and PrPC is not. PrPC and PrPSc contain more methionines than would be expected of a mammalian protein. Furthermore, methionines are easily oxidized by chemicals. The extent to which a methionine can be oxidized is dependent on its surface exposure. The more exposed a methionine is the more easily it can be oxidized. Measuring the extent of methionine can be used to map the surface of PrPC and PrPSc. Digestion of PrPC or PrPSc with an enzyme (trypsin) yields a set of protein fragments called peptides. We identified 11 different methionine-containing peptides that account for every methionine present in PrPC from bank vole, mouse, sheep, deer, and elk. We showed that we could measure the amount of the oxidized and unoxidized forms of these peptides. If a sample is oxidized, then this information allows us to determine the surface exposure of each methionine in PrPC or PrPSc.

Technical Abstract: The normal cellular prion protein (PrPC) and its infectious conformer, PrPSc, possess a disproportionately greater amount of methionines than would be expected for a typical mammalian protein. A variety of oxidants have been used to oxidize the thioether of methionine to the corresponding sulfoxide. Oxidation of methionine can be used to map the surface of a protein. 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 suitable for a multiple reaction monitoring (MRM)-based analysis. The optimal trapping cartridge loading time was determined to ensure that the peptide and its sulfoxide analogs would be retained. The optimal time for the shorter peptides (TNMK, ENMNR, ENMYR, IMER, MMER, MIER) was determined to be 3 minutes. Five minutes was determined to be optimal for the longer peptides (MLGSAMSR, LLGSAMSR, PMIHFGNDWEDR, VVEQMCVTQYQK, and VVEQMCITQYQR). This approach can be used to determine the surface exposure of each methionine by measuring its oxidation after reaction with added hydrogen peroxide.