Enzymatic synthesis of isotopically labeled hydrogen peroxide for mass spectrometry-based applications

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 sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (H218O2) can be used to block unoxidized methionines and enable accurate measurement of in vivo levels of methionine oxidation. However, H218O2 is an expensive reagent that can be difficult to obtain from commercial sources. Here, we report a method for synthesizing H218O2 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 H218O2 from 18O2 and assessed its concentration, purity, and utility in measuring methionine oxidation levels by mass spectrometry. ### Competing Interest Statement The authors have declared no competing interest.