PrPSc and PrPCare isoforms, since they possess identical covalent structures and identical post-translational modifications. The same amino acid may react differently with the same chemical reagent in an isoform-dependent manner. The site of covalent modification can be identified by mass spectrometry or by Western blot, if the epitope of the primary antibody contains an amino acid that can be covalently modified by a selected reagent.We synthesized a number of small molecules and reacted them with PrPSc, PrPC, and recombinant PrP. These reagents preferentially react with the e-amino group of lysine. The reaction mixtures were analyzed by mass spectrometry and Western blot. The antibodies recognize a lysine-containing epitope that is encrypted in the PrPSc isoform, but exposed in the PrPCisoform.These reagents covalently modify lysine residues, so they block the recognition of PrPC by the antibodies. In this way Western blot analysis of these reactions permits the detection of prion infected brain extracts without the need for proteinase K digestion. This is important, because although proteinase K-resistant PrP is diagnostic for disease, much of infectious PrP is proteinase K-sensitive. In addition these reagents can be used, with an appropriate antibody, to determine which amino acids of PrPSc are exposed on the surface and which are encrypted, thus providing useful structural information. This approach was used to distinguish among strains of hamster-adapted scrapie without the use of proteinase K. Mass spectrometry-based analysis was used to quantitate these differences and to analyze the relative reactivity of the various lysines present in hamster PrP.

Small molecules and antibodies: A means of distinguishing between PrP<sup>C</sup> and PrP<sup>Sc</sup>

248th National Meeting (2014)

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Small molecules and antibodies: A means of distinguishing between PrPC and PrPSc

Small molecules and antibodies: A means of distinguishing between PrP^C and PrP^Sc