Detecting Differences in Prion Protein Conformation by Quantifying Methionine Oxidation

Abstract

A prion's pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion's conformation and to distinguish among prion strains, which is an important component of food safety.

General Method of Quantifying the Extent of Methionine Oxidation in the Prion Protein

“…However, after 15 min of chymotryptic digestion, an observed difference (p < 0.04) between the extent of oxidation of the methionines in the peptide was seen, indicating that chymotrypsin preferentially cleaves the unoxidized methionine. 41 Cervids possess a polymorphism at position 132, where the methionine is replaced by leucine. The peptide, LLGSAMSR, is produced by the tryptic digestion of elk PrP.…”

“…We have observed that chymotrypsin preferentially cleaves at the carboxyl terminus of methionine over that of the sulfoxide analogue. 41 Thus, an extended chymotrypsin digestion can skew the proportion of oxidized methionine in a sample.…”

“…Previous work has shown that the extent of methionine oxidation in hamster PrP, after treatment with hydrogen peroxide, was consistent with the solution NMR structure. 40,41 Since the structures of mouse, bank vole, sheep, and cervid PrP have been determined, 53−56 the empirical results of this MRM-based method can be accurately compared to known rPrP structures. Hydrogen peroxide based oxidation of prions does not substantially alter the infectivity of prions, 43,47 suggesting that such oxidation does not substantially perturb the prion structure.…”

“…40 An alternate approach employed trypsin digestion and MRM-based analysis to quantify the extent of methionine oxidation. 41 This MRM-based approach permitted the analysis of a more complete set of methionines. Furthermore, the MRM-based approach requires substantially smaller amounts of sample.…”

Distinguishing post‐translational modifications in dominantly inherited frontotemporal dementias: FTLD‐TDP Type A (GRN) vs Type B (C9orf72)

“…Several of these are modifications known to occur during sample processing (e.g., oxidation) or electrospray ionisation MS (e.g., water loss, ammonia loss and metal adducts) [32]. However, although oxidation is sometimes associated with sampling processing, it is impossible to rule out that some sites that have been reported previously may have a biological significance [6,23,33]. For this reason, this modification has been included in our analysis.…”