Prions are the etiological agents of transmissible spongiform encephalopathies (TSEs), a class of fatal neurodegenerative diseases affecting humans and other mammals. The pathogenic prion protein is a misfolded form of the host-encoded prion protein and represents the predominant, if not sole, component of the infectious agent. Environmental routes of TSE transmission are implicated in epizootics of sheep scrapie and chronic wasting disease (CWD) of deer, elk, and moose. Soil represents a plausible environmental reservoir of scrapie and CWD agents, which can persist in the environment for years. Attachment to soil particles likely influences the persistence and infectivity of prions in the environment. Effective methods to inactivate TSE agents in soil are currently lacking, and the effects of natural degradation mechanisms on TSE infectivity are largely unknown. An improved understanding of the processes affecting the mobility, persistence, and bioavailability of prions in soil is needed for the management of TSE-contaminated environments.

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiological agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at sub-femtomole levels while animal bioassays, cell culture, and in vitro conversion assays offer ultrasensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with sub-femtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report of the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Prions, the etiological agents in transmissible spongiform encephalopathies, exhibit remarkable resistance to most methods of inactivation that are effective against conventional pathogens. Prions are composed of pathogenic conformers of the prion protein (PrPTSE). Some prion diseases are transmitted, in part, through environmental routes. The recalcitrance of prions to inactivation may lead to a persistent reservoir of infectivity that contributes to the environmental maintenance of epizootics. At present, few methods exist to remediate prion-contaminated lands. Here we examined the ability of peroxymonosulfate to degrade PrPTSE as an initial step toward developing an in situ chemical oxidation process to inactivate prions. We find that peroxymonosulfate rapidly degrades PrPTSE from two species. Transition metal-catalyzed decomposition of peroxymonosulfate to produce sulfate radicals appears to enhance degradation. We further demonstrate that exposure to peroxymonosulfate significantly reduced PrPC-to-PrPTSE converting ability as measured by protein misfolding cyclic amplification, used as a proxy for infectivity. Liquid chromatography-tandem mass spectrometry revealed that exposure to peroxymonosulfate results in oxidative modifications to methionine and tryptophan residues. This study indicates that peroxymonosulfate may hold promise for in situ remediation of prion-contaminated surfaces.

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