NR ATKH
AU Gielbert,J.; Clark,J.K.; Docherty,J.G.; Gill,A.; Sauer,M.J.
TI TSE straintyping by N-terminal sequencing of PrPres using mass spectrometry
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-19
PT Konferenz-Poster
AB
Biochemical straintyping of TSEs has gained increased importance in face of the need to monitor for the possible presence of BSE in sheep, which may be masked by scrapie. 1D western blotting is a well established method enabling discrimination between BSE and scrapie in sheep [1], though its relatively low resolution limits detailed examination of PrPres isoforms.
We have previously used nano-liquid chromatography with ion-trap mass spectrometry to evaluate brain PrP from mice inoculated with various TSE strains. These studies indicated that the ratio of tryptic peptides cleaved from the N-terminal domain of PrPres varies between strains, reflecting differences in accessibility of PrPsc to proteinase K [2].
We have now extended our straintyping studies to sheep TSEs. The approach has been adapted to provide more specific and robust identification and quantitation of N-terminal tryptic peptides of PrPres. A triple quadrupole mass spectrometer has been applied to peptide detection using selected ion monitoring (SIM) and multiple reaction monitoring (MRM). The latter is a well-known technique enabling sensitive and selective detection and quantitation of, for instance, pharmaceutical compounds.
We will describe the following:
1. Application of the synthetic analogues of tryptic peptides to facilitate robust PrPres characterisation.
2. Fragmentation of the synthetic peptides for identification of N-terminal PrPres tryptic peptides.
3. Relative sensitivity and specificity of SIM and MRM approaches for analysis of N-terminal tryptic peptides of PrPres.
4. Application of the LC-MS to systematically evaluate purification methods of PrPres from pooled sheep brain.
References:
[1] Stack, M.J.; Chaplin, M.J.; Clark J.; Acta Neuropathol. 2002 104(3):279-86.
[2] Howells, L Anderson, S, Coldham, N and Sauer, M (2004). Differential diagnosis of TSE: Characterisation and identification of PrP strains by mass spectrometry. Joint Funders' TSE Worksho
AD Janine Gielbert, Jemma K. Clark, John G. Docherty, Maurice Sauer, Veterinary Laboratories Agency, UK; Andrew Gill, Institute for Animal Heralth, UK
SP englisch
PO Deutschland