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 the sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (H₂¹⁸O₂) can be used to block unoxidized methionines and enables 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.