High-resolution differentiation of transmissible spongiform encephalopathy strains by quantitative N-terminal amino acid profiling (N-TAAP) of PK-digested abnormal prion protein

New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease-resistant prion protein (PrP ^Sc) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease-resistant products (PrP ^res) with partially variable N-termini. The conformation(s) of PrP ^sc and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrP ^res gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrP ^res N-terminal tryptic peptides by MS and thus to define the N-terminal amino acid profiles (N-TAAPs) of PrP ^res characteristic for various TSEs in sheep. The fragmentation behaviour of the N-terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrP ^res preparation methods were evaluated and the most effective approach applied to differentiate the N-TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N-TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE-strain specific diagnosis.