Hoare Margaret, Tan Ruiyue, Militi Isabella, Welle Kevin A, Swovick Kyle, Hryhorenko Jennifer R, Ghaemmaghami Sina
Department of Biology, University of Rochester, Rochester, New York 14627, United States.
University of Rochester Mass Spectrometry Resource Laboratory, University of Rochester Medical Center, Rochester, New York 14627, United States.
J Am Soc Mass Spectrom. 2024 Dec 4;35(12):3308-3312. doi: 10.1021/jasms.4c00326. Epub 2024 Oct 4.
Methionine oxidation is involved in multiple biological processes including protein misfolding and enzyme regulation. However, it is often challenging to measure levels of methionine oxidation by mass spectrometry, in part due to the prevalence of artifactual oxidation that occurs during the sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (HO) can be used to block unoxidized methionines and enables accurate measurement of levels of methionine oxidation. However, HO is an expensive reagent that can be difficult to obtain from commercial sources. Here, we report a method for synthesizing HO in-house. Glucose oxidase catalyzes the oxidation of β-d-glucose and produces hydrogen peroxide in the process. We took advantage of this reaction to enzymatically synthesize HO from O and assessed its concentration, purity, and utility in measuring methionine oxidation levels by mass spectrometry.
甲硫氨酸氧化参与多种生物过程，包括蛋白质错误折叠和酶调节。然而，通过质谱法测量甲硫氨酸氧化水平往往具有挑战性，部分原因是在典型蛋白质组学工作流程的样品制备和电离步骤中会出现人为氧化的情况。同位素标记的过氧化氢（HO）可用于阻断未氧化的甲硫氨酸，并能准确测量甲硫氨酸氧化水平。然而，HO是一种昂贵的试剂，很难从商业渠道获得。在此，我们报告一种在内部合成HO的方法。葡萄糖氧化酶催化β-d-葡萄糖的氧化，并在此过程中产生过氧化氢。我们利用这一反应从O酶促合成HO，并评估了其浓度、纯度以及在通过质谱法测量甲硫氨酸氧化水平方面的效用。
