Absolute and relative quantification of sheep brain prion protein (PrP) allelic variants by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation in the tissues of affected individuals of an abnormal form (PrP(Sc)) of a protein naturally produced by the host, the cellular prion protein (PrP(C)). In sheep, susceptibility to TSEs is tightly controlled by polymorphism at positions 136 (A or V), 154 (R or H) and 171 (R or Q) of the Prnp gene encoding the prion protein (PrP). Quantification of PrP variants at positions 136, 154 and 171 can be achieved by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of the respective peptides 114-139, 152-159 and 160-171 obtained after tryptic digestion of the PrP protein. In this study we 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 (18)O-labelled peptide obtained by hydrolysing known amounts of recombinant protein with trypsin in H(2) (18)O. After purification of PrP(C) and PrP(Sc) from the brain of two heterozygous sheep carrying either the ARQ/VRQ or ARR/VRQ genotypes, the proportion of each variant was measured. In the ARQ/VRQ animal, while both variants were equally represented in the normal isoform, the VRQ variant was predominantly found in the abnormal PrP protein, suggesting dissimilar behaviour of the two variants in the pathological process. The situation was even more contrasted in the ARR/VRQ animal where PrP(Sc) was solely composed of the VRQ variant. These two examples clearly illustrate the value of MALDI-TOF analysis, combined with appropriate immunopurification techniques, in seeking a precise understanding of the influence of PrP polymorphisms on TSE pathogenesis.
Morel
Nathalie
N
Commissariat à l'Energie Atomique, iBiTecS, Service de Pharmacologie et d'Immunoanalyse, bâtiment 136, CEA/Saclay, Gif sur Yvette, France. nathalie.morel@cea.fr
Andréoletti
Olivier
O
Grassi
Jacques
J
Clément
Gilles
G
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000821:virology; D001923:Brain Chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D020641:Polymorphism, Single Nucleotide; D018621:PrPC Proteins / Q000032:analysis* / Q000235:genetics; D018620:PrPSc Proteins / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins; D012756:Sheep; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D018621:PrPC Proteins; D018620:PrPSc Proteins; D011994:Recombinant Proteins