Ovalbumin oxidative modification fingerprints depend on gas plasma-driven reactive species profiles

OBJECTIVE: Oxidative protein modifications have been linked to several diseases, but the variety and diversity of modifications are less studied. METHODS: We used the chicken egg protein ovalbumin and gas plasma technology, a potent source of various reactive species, for protein oxidation. Using high-resolution mass spectrometry and an in-house workflow, over 80 distinct oxidative protein modifications were mapped at per-amino-acid resolution. To examine how modification profiles depend on changes in reactive species types and concentrations, we generated 12 distinct argon gas plasmas by systematically varying molecular gas admixtures (water, ethanol, oxygen, and nitrogen). RESULTS: Optical emission spectroscopy (OES) and photometric determination of deposited long-lived species (hydrogen peroxide, nitrite, and nitrate) were applied to profile gas plasma conditions, revealing the admixture-dependent impact on the reactive oxygen/nitrogen species (ROS/RNS) fingerprint. Correlation analysis with mass spectrometry data revealed the significant involvement of atomic oxygen and hydrogen peroxide in protein oxidation. The enrichment of specific reactive species created by a defined gas plasma composition generated specific ovalbumin oxidation profiles resolved per amino acid. Feed gas-dependent oxidation hotspots, such as Trp149 for dry argon gas or Met274 for hydroxyl radical-rich humidified argon gas, were identified. DISCUSSION: This first-of-its-kind study reveals intricate relationships between dynamic reactive species environments and protein oxidation profiles using ovalbumin as a model system.