|Silva, Christopher - Chris
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2012
Publication Date: 8/22/2012
Citation: Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson-Beltran, M.L., Hui, C., Carter, J.M. 2012. Detecting and discriminating among pathogenic protein conformers(prions), using mass spectrometry-based and antibody-based approaches(Abstract). Meeting Abstract. Poster 216.
Technical Abstract: A set of fatal neurological diseases that includes scrapie and chronic wasting disease (CWD) are caused by a pathological protein referred to as a prion (PrPSc). A prion propagates an infection by converting a normal cellular protein (PrPC) into a prion. Unlike viral, bacterial, or fungal pathogens, the information necessary to propagate the prion is contained solely in the conformation of the prion isoform and not in nucleic acids. For a given host there is more than one possible prion phenotype or strain, so any analytical method must not only be able to detect a prion but also to discriminate among the known strains. Two approaches to this problem will be discussed. In the first we employ a nano LC-MS-MS system to quantitate the characteristic tryptic peptides from prions using the multiple reaction monitoring method (MRM) and stable-isotope labeled internal standards. Our limit of detection is in the attomole range (10-18 mole). This approach can be used to detect a variety of prion strains. The chemical environment of an amino acid present in PrP is conformation (PrPSc or PrPC) dependent; in the second approach, we exploit this property and use small molecules to covalently modify the PrPC isoform in the presence of the PrPSc isoform. We selected reagents that covalently modify the amino the acid side chains present in epitopes recognized by commercially available anti-PrP antibodies and were able to detect conformation-dependent differences in reactivity by simply probing a Western blot of the reaction mixture with one of those antibodies. In this way, a combination of synthetic reagents and Western blot based analysis can be used to detect the presence of prions and to distinguish among prion strains.