Pyroglutamyl-N-terminal prion protein fragments in sheep brain following the development of transmissible spongiform encephalopathies

Protein misfolding, protein aggregation and disruption to cellular proteostasis are key processes in the propagation of disease and, in some progressive neurodegenerative diseases of the central nervous system, the misfolded protein can act as a self-replicating template or prion converting its normal isoform into a misfolded copy of itself. We have investigated the sheep transmissible spongiform encephalopathy, scrapie, and developed a multiple selected reaction monitoring (mSRM) mass spectrometry assay to quantify brain peptides representing the ragged N-terminus and the core of ovine prion protein (PrPSc) by using Q-Tof mass spectrometry. This allowed us to identify pyroglutamylated N-terminal fragments of PrPSc at residues 86, 95 and 101, and establish that these fragments were likely to be the result of in vivo processes. We found that the ratios of pyroglutamylated PrPSc fragments were different in sheep of different breeds and geographical origin, and our expanded ovine PrPSc assay was able to determine the ratio and allotypes of PrP accumulating in diseased brain of PrP heterozygous sheep; it also revealed significant differences between N-terminal amino acid profiles (N-TAAPs) in other types of ovine prion disease, CH1641 scrapie and ovine BSE. Variable rates of PrP misfolding, aggregation and degradation are the likely basis for phenotypic (or strain) differences in prion-affected animals and our mass spectrometry-based approach allows the simultaneous investigation of factors such as post-translational modification (pyroglutamyl formation), conformation (by N-TAAP analysis) and amino-acid polymorphisms (allotype ratio) which affect the kinetics of these proteostatic processes

Inactivation of H-type and L-type bovine spongiform encephalopathy following recommended autoclave decontamination procedures

The resistance of H-type and L-type BSE prions to autoclaving under EU regulation conditions for specified risk material is unknown. We employed transgenic mouse (bovinized line tg1896) bioassay to assess the efficacy of such decontamination on L- and H-type BSE. For each source, titre calculation was based on the comparison of incubation period and attack rate prior to and after decontamination. The infectious titre of L-type BSE was reduced by at least 9.40 log10 and of H-type BSE by at least 3.94 log10. In fact, no infectivity was detected for L-type or H-type BSE post-inactivation even at a 10–1 dilution.</p