Location: Produce Safety and Microbiology ResearchTitle: Using targeted mass spectrometry, covalent modification, Western blot, and bioassay to quantitate the relative role of specific lysines in prion replication
Submitted to: American Chemical Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/9/2020
Publication Date: 8/17/2020
Citation: C.J. Silva, M.L. Erickson-Beltran, I.C. Dynin. 2020. ANYL 10: Using targeted mass spectrometry, covalent modification, Western blot, and bioassay to quantitate the relative role of specific lysines in prion replication. American Chemical Society Abstracts. ACS Fall 2020 Virtual Meeting & Exposition: 10-ANYL.
Technical Abstract: Prions (PrPSc) are the simplest pathogens. They consist of refolded multimers of a single natively expressed normal cellular prion protein (PrPC) and propagate by inducing PrPC to adopt the PrPSc conformation. The information necessary to effect this transformation resides solely in the conformation of PrPSc. The epsilon-amino group of lysine in hamster-adapted scrapie was acetylated by either acetic anhydride (Ac2O) or the N-hydroxysuccinimide ester of acetic acid (Ac-NHS). Targeted mass spectrometry or Western blot-based analysis was used to quantitate the extent of each lysine’s acetylation. In parallel, the loss of infectivity associated with lysine acetylation was quantitated using the incubation time bioassay. These results indicate that at the highest reagent concentration, infectivity was reduced by ten-fold. Ten of the eleven prion lysines were acetylated (25-400-fold) to a greater than ten-fold extent. Only one lysine (K220) had a reactivity that correlated with the loss of infectivity. These covalent modifications do not substantially perturb the prion structure, as measured by bioassay, so they can be used to infer structural information about prions. In principle, this approach can be used to clarify the role of other amino acids involved in prion replication, such as methionine.