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Title: Utility of mass spectrometry in the diagnosis of prion diseases

Author
item Silva, Christopher - Chris
item ONISKO, BRUCE - Former ARS Employee
item Dynin, Irina
item Erickson-Beltran, Melissa
item REQUENA, JESUS - University Of Santiago De Compostela
item Carter, John

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2011
Publication Date: 2/2/2011
Citation: Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.J., Carter, J.M. 2011. Utility of mass spectrometry in the diagnosis of prion diseases. Analytical Chemistry. 83(5):1609-1615. doi:10.1021/ac102527w.

Interpretive Summary: Early detection of prion diseases, such as scrapie in sheep, depends on very sensitive detection of very small amounts of the specific misfolded proteins that make up prions. We have developed a method for such detection, using a mass spectrometer instrument, which can identify prion fragments. In this manuscript we show how this method also can be used to measure the amount of infectious prions present in a number of animals. To do this we used chemistry to make the same fragment molecules as are produced in disease, and we measured them as a standard. We were able to measure amounts as low as 100 attomoles (1 x 10-18 moles), which is far below the limit of detection in a microscope. We used this method to analyze prions in the brains of hamsters and mice. We could detect prions five weeks after the animals were experimentally infected with prions, which is long before they began to show symptoms of the disease. We also used this method to detect prions in the brains of sheep that were naturally infected with scrapie. The early diagnosis this method provides can be used to help in management of flocks, to prevent spread of prion disease in livestock.

Technical Abstract: We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the selected analyte peptides and their oxidized analogs to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 attomoles. Non-analyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice five weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or three mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the non-obex/non-anterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.