NR AXNC
AU Gielbert,A.; Ho,S.M.; Davis,L.A.; Gill,A.C.; Sauer,M.J.
TI High Resolution TSE Straintyping by LC-MS/MS - Distinctions Between Ovine BSE, Classical Scrapie and Experimental Scrapie Isolates
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.58
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: As new forms of TSE continue to be identified, their differential diagnosis becomes increasingly important. Biochemical screening tests can differentiate TSEs, making use of differing structural properties of PrPsc. BSE and classical scrapie can be distinguished by Western blotting (WB): proteinase K (PK) digested PrPsc (PrP27-30) reveals a characteristic shift of the unglycosylated band to lower molecular weight, attributed to a significant difference in PK cleavage sites. Determining the N-terminal amino acid profile (N-TAAP) of PrP27-30 in detail, utilizing a high resolution method such as mass spectrometry (MS), would also allow differentiation between TSEs where differences in PK cleavage sites are more subtle.
Objective: Utilizing the high resolution provided by MS detection, we aim to investigate the potential of high resolution N-TAAP profiling of PrP27-30 for differential diagnosis of TSEs.
Methods: Brain material from sheep infected naturally or experimentally with TSE was homogenized, digested by PK, and subjected to sodium phosphotungstic acid (NaPTA) precipitation. N-TAAP analysis was performed by quantification of peptides formed upon trypsinolysis of PrP27-30 under denaturing conditions. A MS method provided peptide quantification by reference to synthetic standards of nine N-terminal PK fragments and three C-terminal tryptic fragments. This allowed both the quantitative evaluation of the N-TAAP of PrP27-30 and normalisation against the total PrP 27-30 isolated by reference to the concentration of the 'conserved' C-terminal peptides.
Results: The dominant PK cleavage sites for classical scrapie were at W84, H88 and W93 and contrasted with those for ovine BSE which were at W93, Q95, H99, Q101 and W103. The ratio of the total quantity of N-terminal peptides to the C-terminus was somewhat higher for scrapie compared to that for BSE, suggesting that there may be further cleavage points for BSE which have not yet been identified. More subtle differences were also observed, for instance between field case samples of different genotypes and samples derived from experimental SSBP/1 inoculations.
Conclusions: Distinctive differences in N-TAAP of PrP27-30 between strains, and a preliminary indication of subtle differences in structures between genotypes and scrapie isolates were identified. High resolution N-TAAP profiling is a powerful means to differentiate TSEs.
AD A. Gielbert, S.M. Ho, L.A. Davis, M.J. Sauer, Veterinary Laboratories Agency, Molecular Pathogenesis and Genetics, UK; A.C. Gill, Roslin Institute, Neuropathogenesis Unit, UK
SP englisch
PO Schottland