Silva Christopher J, Erickson Beltran Melissa L, Requena Jesús R
Produce Safety and Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, United States.
CIMUS Biomedical Research Institute & Department of Medical Sciences, University of Santiago de Compostela-IDIS, Santiago de Compostela 15782, Spain.
ACS Omega. 2025 Jan 3;10(1):1320-1330. doi: 10.1021/acsomega.4c08892. eCollection 2025 Jan 14.
Scrapie is a prion disease of sheep and goats. Prions (PrP) replicate by inducing a natively expressed protein (PrP) to refold into the prion conformation. PrP and PrP contain a disproportionately large number of methionines. Surface exposed methionines are more prone to chemical oxidation. Chemical oxidation is a means of measuring the surface exposure of the methionines in a prion, as these covalent changes are retained after an oxidized prion is denatured prior to analysis. Scrapie prions and recombinant sheep prion protein were oxidized in 0, 10, 20, or 50 mM solutions of hydrogen peroxide. The samples were digested with trypsin or trypsin followed by chymotrypsin to yield a set of peptides (TNMK, MLGSAMSR, ENMYR, IMER, VVEQMCITQYQR) containing the methionines present in sheep PrP. The mass spectrometry based multiple reaction monitoring (MRM) method was used to analyze these peptides. Analysis of the rPrP samples showed that surface exposed methionines (132, 137, and 157) were more oxidized than those less surface exposed (209 and 216). The extent of methionine oxidation in sheep scrapie PrP is 216 > 137 > 132 > 157 > 209 > 112. These results demonstrate that this approach can be used to map the surface exposure of the methionines in order to distinguish among PrP conformations and effect a kind of conformational sequence.
羊瘙痒病是绵羊和山羊的一种朊病毒病。朊病毒（PrP）通过诱导天然表达的蛋白质（PrP）重新折叠成朊病毒构象来进行复制。PrP和PrP含有数量不成比例的大量甲硫氨酸。表面暴露的甲硫氨酸更容易发生化学氧化。化学氧化是一种测量朊病毒中甲硫氨酸表面暴露程度的方法，因为这些共价变化在氧化的朊病毒在分析前变性后仍会保留。羊瘙痒病朊病毒和重组绵羊朊病毒蛋白在0、10、20或50 mM的过氧化氢溶液中进行氧化。样品用胰蛋白酶或胰蛋白酶随后用糜蛋白酶消化，以产生一组包含绵羊PrP中存在的甲硫氨酸的肽段（TNMK、MLGSAMSR、ENMYR、IMER、VVEQMCITQYQR）。基于质谱仪的多反应监测（MRM）方法用于分析这些肽段。对rPrP样品的分析表明，表面暴露的甲硫氨酸（132、137和157）比表面暴露较少的甲硫氨酸（209和216）氧化程度更高。绵羊瘙痒病PrP中甲硫氨酸的氧化程度为216 > 137 > 132 > 157 > 209 > 112。这些结果表明，这种方法可用于绘制甲硫氨酸的表面暴露情况，以区分PrP构象并实现一种构象序列。
