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.
J Am Soc Mass Spectrom. 2023 Feb 1;34(2):255-263. doi: 10.1021/jasms.2c00280. Epub 2023 Jan 6.
The normal cellular prion protein (PrP) and its infectious conformer, PrP, possess a disproportionately greater amount of methionines than would be expected for a typical mammalian protein. The thioether of methionine can be readily oxidized to the corresponding sulfoxide, which means that oxidation of methionine can be used to map the surface of the conformation of PrP or PrP, as covalent changes are retained after denaturation. We identified a set of peptides (TNK, LGSASR, LLGSASR, PIHFGNDWEDR, ENNR, ENYR, IER, ER, IER, VVEQCVTQYQK, and VVEQCITQYQR) that contains every methionine in sheep, cervid, mouse, and bank vole PrP. Each is the product of a tryptic digestion and is suitable for a multiple reaction monitoring (MRM) based analysis. The peptides chromatograph well. The oxidized and unoxidized peptides containing one methionine readily separate. The unoxidized, two singly oxidized, and doubly oxidized forms of the LGSASR and ER peptides are also readily distinguishable. This approach can be used to determine the surface exposure of each methionine by measuring its oxidation after reaction with added hydrogen peroxide.
正常细胞朊病毒蛋白（PrP）及其感染性构象 PrP 比典型的哺乳动物蛋白具有更多的蛋氨酸。蛋氨酸的硫醚可以很容易地氧化为相应的亚砜，这意味着蛋氨酸的氧化可以用于绘制 PrP 或 PrP 构象的表面，因为在变性后共价变化得以保留。我们确定了一组肽（TNK、LGSASR、LLGSASR、PIHFGNDWEDR、ENNR、ENYR、IER、ER、IER、VVEQCVTQYQK 和 VVEQCITQYQR），其中包含绵羊、鹿、小鼠和田鼠 PrP 中的每一个蛋氨酸。每个都是胰蛋白酶消化的产物，适合基于多重反应监测（MRM）的分析。肽的色谱分离效果很好。含有一个蛋氨酸的氧化和未氧化肽很容易分离。LGSASR 和 ER 肽的未氧化、单氧化和双氧化形式也很容易区分。这种方法可以通过测量与外加过氧化氢反应后的氧化程度来确定每个蛋氨酸的表面暴露情况。
