NR AXTA
AU Morel,N.; Andreoletti,O.; Grassi,J.; Clement,G.
TI Absolute and Relative Quantification of Sheep Brain Prion Protein (prp) Variants by Maldi-Tof Mass Spectrometry
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.06
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Transmissible Spongiform Encephalopathies (TSE) are characterized by the accumulation in tissue of affected individuals of an abnormal form of a protein naturally produced by the host, the prion protein, PrP. through post-translational modifications which induce a conformational change. In sheep, the susceptibility to TSE is tightly controlled by the polymorphism at position 136 (A or V), 154 (R or H) and 171 (R or Q) of the Prnp gene encoding for the sheep prion protein (PrP). Quantification of PrP variants at position 136, 154 and 171 can be achieved by MALDI-TOF massspectrometry analysis of protein fragments, respectively peptides 114-139 and 152159 and the 160-171, obtained after tryptic digestion of the PrP protein.
In this study we monitored and quantified the tryptic peptide 114-139 containing the first polymorphic site. Quantification was either relative, between variants of this peptide, or absolute with respect to the C-terminally 18O labelled peptide obtained by hydrolyzing known amounts (quantified by UV-absorption) of recombinant protein with trypsin in H218O.
After extraction and immunopurification of PrPc and PrPsc from brains taken from scrapie infected heterozygote animals ARQ/VRQ or VRQ/ARR, each variants proportions was measured using this method. In ARQ/VRQ animals, while both variant were equally represented in the normal isoform of the protein, VRQ variant was predominantly found in the abnormal PrP protein, suggesting a dissimilar behaviour of both variant in the pathological process. The situation was even more contrasted in VRQ/ARR animals where PrPsc was solely composed of the VRQ variant.
These two examples clearly illustrate the interest of the MALDI -TOF analysis combined with appropriate immunopurification techniques for a precise understanding of the influence of the PrP polymorphism in TSE pathogenesis.
AD N. Morel, J. Grassi, CEA, Service de Pharmacologie et d'Immunoanalyse, France; O. Andréoletti, Ecole Nationale Vétérinaire de Toulouse, Umr Inra Envt, France; G. Clément, Inra, Laboratoire d'Immuno-Allergie Alimentaire, France
SP englisch
PO Schottland