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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AGENTS IN LIVESTOCK, WILDLIFE, AGRICULTURAL PRODUCTS, AND THE ENVIRONMENT Title: Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation

Authors
item SILVA, CHRISTOPHER
item Dynin, Irina
item ERICKSON-BELTRAN, MELISSA
item Requena, Jesus -
item Balachandran, Aru -
item Onisko, Bruce
item Hui, Colleen
item CARTER, JOHN

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2013
Publication Date: March 4, 2013
Repository URL: http://pubs.acs.org/doi/pdf/10.1021/bi3016795
Citation: Silva, C.J., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R., Balachandran, A., Onisko, B.C., Hui, C., Carter, J.M. 2013. Using mass spectrometry to detect prions and oxidized prions in scrapie-infected sheep and CWD-infected elk. Biochemistry. 52:2139-2147. doi:10.1021/bi3016795.

Interpretive Summary: We developed a sensitive method (mass spectrometry) of detecting a disease agent (prion disease) in sheep and elk. This method represents an improvement over the standard tests, since it requires much less tissue and is more sensitive. In addition, we used this method to detect the amount of oxygen that was combining with the disease agent. We found that very little oxygen combined with the disease agent, even though this agent was very susceptible to combination with oxygen. This means that these disease agents are shielded from oxygen and do not seek other oxygen-containing molecules when they replicate.

Technical Abstract: We developed a sensitive mass spectrometry-based method of quantitating the prions present in elk and sheep. Calibration curves relating the area ratios of the selected analyte peptides and their homologous stable isotope labeled internal standards were prepared. This method was compared to the ELISA, Western blot, and immunohistochemistry methods. Mass spectrometry could detect the presence of prions in all samples and required much lower amounts of tissue(600 'g v. 120 mg), even though the tissue was non-obex brain. In addition to the analyte peptide (VVEQMCITQYQR), additional peptides GENFTETDIK(sheep and elk), ESQAYYQR (sheep), or ESEAYYQR (elk) were used to confirm the presence of prions in the samples. The ratio of oxidized to the unoxidized methionine at position 216 was determined to be comparable to that previously reported. These results were consistent with MetSO216 not being actively recruited and converted to prions as has been speculated before. Two unnatural polymorphisms (I218V and I218T) of the sheep Prnp gene were prepared. The purified proteins were used to determine that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 in the order, I>V>T. This indicates that the presence of isoleucine in sheep, deer, elk, and human PrP makes that methionine inherently more susceptible to oxidation than the analogous methionine in hamster PrP. The levels of oxidized methionine in sheep and elk prions indicate that the oxidized form is not preferentially recruited and converted to the prion isoform.

Last Modified: 8/19/2014
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