Enzymatic Synthesis\nof Isotopically Labeled Hydrogen\nPeroxide for Mass Spectrometry-Based Applications

Methionine oxidation is involved in multiple biological\nprocesses\nincluding protein misfolding and enzyme regulation. However, it is\noften challenging to measure levels of methionine oxidation by mass\nspectrometry, in part due to the prevalence of artifactual oxidation\nthat occurs during the sample preparation and ionization steps of\ntypical proteomic workflows. Isotopically labeled hydrogen peroxide\n(H<sub>2</sub><sup>18</sup>O<sub>2</sub>) can be used to block unoxidized\nmethionines and enables accurate measurement of <i>in vivo</i> levels of methionine oxidation. However, H<sub>2</sub><sup>18</sup>O<sub>2</sub> is an expensive reagent that can be difficult to obtain\nfrom commercial sources. Here, we report a method for synthesizing\nH<sub>2</sub><sup>18</sup>O<sub>2</sub> in-house. Glucose oxidase\ncatalyzes the oxidation of β-d-glucose and produces\nhydrogen peroxide in the process. We took advantage of this reaction\nto enzymatically synthesize H<sub>2</sub><sup>18</sup>O<sub>2</sub> from <sup>18</sup>O<sub>2</sub> and assessed its concentration,\npurity, and utility in measuring methionine oxidation levels by mass\nspectrometry.