Amino Acid, Peptide, and Protein Level Mapping of Lysozyme Oxidation by Ozone-Dominated Nonthermal Plasma

Abstract Chicken lysozyme C was treated with atmospheric pressure nonthermal plasma and was analyzed at amino acid, peptide and protein level. Hydrolysis conditions were optimized to retain tryptophan and selected oxidation products, with 6 M HCl containing 0.2% tryptamine under O2-deprived conditions providing the best results. Plasma treatment significantly decreased tryptophan, histidine and tyrosine, while other amino acids were minimally affected. Methionine remained unaffected despite its known susceptibility to oxidation. Tryptophan oxidation products kynurenine/N′-formylkynurenine (quantified as kynurenine) and oxindolylalanine increased. Peptide level analysis showed significant increases in several oxidized peptide forms, predominantly involving solvent accessible residues, including tryptophan modifications assigned to W[+4] (kynurenine/N′-formylkynurenine), W[+20] (hydroxykynurenine) and W[+14] (oxidation and dehydrogenation) and conversion of histidine to asparagine. Protein level analysis with SDS-PAGE revealed both reducible and reduction-stable oligomers, consistent with disulfide-mediated and additional covalent cross-linking, respectively. Lower-molecular-weight bands suggested limited fragmentation. These findings provide insight into protein modifications relevant to plasma-based food decontamination.