Substantial evidence indicates that the disease-associated conformer of the
prion protein (PrP(TSE)) constitutes the etiologic agent in
prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal
prion protein (PrP(C)) into a β-sheet rich form resistant to
proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a
protein's signature
peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature
peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative
protease (
chymotrypsin) can produce signature
peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature
peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature
peptide retaining the same amino acid sequence across most mammals naturally susceptible to
prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic
peptide in a LC-MRM AQUA workflow.