NR AWFK
AU Gielbert,J.; Ho,S.; Gill,A.C.; Sauer,M.J.
TI Straintyping sheep brain PrPsc by liquid chromatography-tandem mass spectrometry
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions PR-14
PT Konferenz-Poster
AB Differential diagnosis of TSEs is particularly important because of the need to distinguish scrapie from possible BSE infection in small ruminants. Until now, routine biochemical screening tests that can differentiate TSEs are based on Western blotting. However, the resolution of these tests is relatively low and known to be unable to differentiate BSE from some scrapie isolates such as CH1641. A strategy has been developed at VLA for high resolution strain differentiation by sequencing the Nterminus of PrP27-30 by mass spectrometry. This approach has been progressed by incorporating quantification of N-terminal fragments against synthetic standards. For this purpose a new quantitative liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method has been developed. PrP27-30 was extracted from brain of naturally infected sheep by PK treatment and precipitation by sodium phosphotungstic acid (NaPTA) and/or centrifugation through a sucrose cushion (SCC). The resulting samples were reduced, alkylated and digested by trypsin under denaturing conditions. The quantities of the various N-terminal tryptic peptides extracted were initially determined for pooled sheep brain. At least 10 picomoles of PrPsc could be extracted from 0.5 g brain of scrapie-infected sheep and that the dominant PK cleavage sites were at W84, H88 and W93. An extraction method which included NaPTA but not SCC proved to give highest recovery of PrP27-30. The outcome will be reported of application of this approach to confirm previous qualitative comparisons of mouse TSE strains. Brain PrP27-30 N-terminus profiles will be described for scrapie field cases (various breeds and genotypes) and for sheep inoculated with TSE isolates (CH1641 and SSBP/1) and BSE
AD J. Gielbert, S. Ho, M.J. Sauer: Veterinary Laboratories Agency, Addlestone, United Kingdom; A.C. Gill: Institute for Animal Health, Compton, United Kingdom
SP englisch
PO Italien