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Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 5/17/2017 Publication Date: 8/23/2017 Citation: Silva, C.J., Erickson-Beltran, M.L. 2017. Detecting and distinguishing among covalent and non-covalent differences in proteins: Shiga toxins and prions. Meeting Abstract. 254: 224-AGFD. Interpretive Summary: The molecules that cause foodborne disease are very different from one another. Some contain fewer than 30 atoms and others contain more than 10,000. The smaller molecules cause disease by reacting with cells in the host. These smaller molecules are destroyed in this process. The much larger protein toxins cause disease by being catalysts, molecules that change other molecules, but remain unaffected. This means that the amount of protein toxins required to cause disease is much less than that of the smaller molecules. Another kind of protein called a prion causes illness as it multiplies. Prions cause disease by converting a normal cellular protein (PrPc) into a prion (PrPsc), which makes more prions and results in disease. Detecting and distinguishing among these molecules is important and also challenging. The E. coli bacteria associated with outbreaks of food poisoning produce a protein toxin called Shiga toxin. These bacteria are referred to as STEC. Minute amounts of Shiga toxins are enough to cause the more serious symptoms of food poisoning. Since there are least ten different kinds of Shiga toxins, it is important to be able to detect small amounts of Shiga toxins and to distinguish among the kinds of these toxins. We will describe a safe method to detect and distinguish among these toxins at low levels. A prion and PrPC are made up of the same number and kinds of atoms and only differ in their shape. We describe a sensitive method to detect and quantify prions in a sample containing PrPC. In this way we can detect the presence of the prion shape in a sample using standard laboratory techniques, such as mass spectrometry or antibody-based detection. Technical Abstract: The structural variety of food associated contaminants is remarkable. This diversity spans compounds from small molecules to protein toxins to infectious proteins. The small molecules are stoichiometric toxins while the proteins are catalytic toxins. This means that the molar amount of a protein toxin required to sicken a person is substantially less than that of a small molecule. Infectious proteins (prions) are able to convert a normal cellular prion protein (PrPC) into a prion (PrPSc), thereby propagating a pathology as they amplify their numbers. Unlike protein toxins, structural differences between the PrPC and PrPSc are solely conformational. Detecting and distinguishing among these foodborne prions and protein toxins is important and also a significant challenge. Shiga toxin is the major virulence factor of STEC (Shiga toxin producing Escherichia coli), a source of many of the serious foodborne disease outbreaks. We have developed a sensitive and specific mass spectrometry-based method of detecting Shiga toxins, based on the detection of characteristic tryptic peptides derived from the non-toxic B subunits of these toxins. An artificial gene encoding a single protein containing the relevant peptides was used to generate the needed 15N-labeled internal standards. This approach can be used to quantify and distinguish among the known type 1 and type 2 Shiga toxins in the low attomole range in complex media, including human serum. Prions can be transmitted by consuming contaminated food. They cause protein misfolding diseases, such as transmissible spongiform encephalopathy (TSE). PrPC is monomeric and sensitive to proteinase K (PK) digestion, while PrPSc is multimeric and relatively resistant to PK. Multiple prion conformations or strains can be derived from the same PrPC substrate, each strain having a distinct TSE pathology. We have developed a mass spectrometry-based method of detecting prions in the attomole range. We use small molecule reagents to distinguish among prion strains and to detect them by mass spectrometry-based or antibody-based methods. The required internal standards are derived from the 15N-labeled recombinant PrP proteins. In this way protein conformations can be detected using either mass spectrometry or antibody-based methods. |
