|Silva, Christopher - Chris|
Submitted to: Journal of American Society for Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2007
Publication Date: 3/28/2007
Citation: Onisko, B.C., Dynin, I.A., Requena, J.R., Silva, C.J., Erickson, M.L., Carter, J.M. 2007. Mass Spectrometric Detection of Attomole Amounts of the Prion Protein by nanoLC-MS-MS. Journal of American Society for Mass Spectrometry. Volume(18); 1070-1079 Interpretive Summary: Prions are the cause of fatal brain diseases such as scrapie in sheep and bovine spongiform encephalopathy (BSE) is cows. Prions have been shown to be present in blood by experimentally transfusing blood from infected animals to uninfected animals. At present there are no reliable methods to diagnose TSEs in live animals. We developed a method to detect prions using nano-scale purification coupled to an extremely sensitive detector (mass spectrometer). This method allows us to detect material at a level at least 10,000 times lower than any currently approved diagnostic test.
Technical Abstract: Quantitation of prions in biological samples other than brain or spinal cord is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases in animal and humans known as Transmissible Spongiform Encephalopathies (TSEs). At present there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30-625 amol/mL. We studied quantitation of the prion protein by use of nano-scale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation and digestion with trypsin of the prion protein. Our attempt to improve this method using derivatization of PrP peptides with phenylisothiocyanate (PITC) was not successful because of decreased ionization efficiency of the PITC-derivatized peptides. The VVEQMCTTQYQK method has an LOD of 20-30 amol, which is at least four orders of magnitude more sensitive than published antibody methods.