A prion’s (PrPSc) distinct pathogenic character is enciphered solely in its conformation. Prions replicate by inducing a natively expressed prion protein (PrPC) to adopt the PrPSc conformation. Although distinguishing between protein conformations is challenging, PrPSc can be purified from PrPC using proteinase K (PK) digestion and ultracentrifugation. The resulting characteristic protein (PrP 27-30) can, after denaturation, be detected by mass spectrometry using the multiple reaction monitoring method (MRM). MRM employs a triple quadrupole mass spectrometer’s mass filters to isolate and detect specific peptides derived from the tryptic digestion of PrP 27-30. We used the MRM method to quantitate the temporal increase of PrP 27-30 in hamsters following a peripheral (intra-peritoneal) challenge using Sc237 prions. PrP 27-30 increased in a log linear relationship over time and was detectable at a time comparable to its earliest detection by bioassay. We adapted our MRM method to detect and quantify the oxidation of a methionine at position 213 (Met213) of the prion protein. Oxidation of Met213 has been proposed as a covalent signature of prions. We quantitated the change in Met213 oxidation over time and demonstrated that it decreased, indicating that its oxidation is not a covalent prion signature. 1.8% of the amino acids in a typical mammalian protein are methionines. In contrast, methionines comprise 4.3% of the amino acids in the hamster prion protein, suggesting the possibility that, in the prion protein, methionines serve another purpose. We optimized the MRM parameters for a set of methionine containing peptides, which allowed us to quantify the extent of methionine oxidation of all nine methionines in the hamster prion protein. After reaction with oxidants, such information can provide invaluable structural information for the prion conformation.

Using mass spectrometry to detect the presence of and dissect the structure of prions

ACS Spring Meeting 2021

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