Lysozyme oxidation by singlet molecular oxygen: Peptide characterization using [¹⁸ O]-labeling oxygen and nLC-MS/MS

Singlet molecular oxygen (¹ O₂ ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for ¹ O₂ , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by ¹ O₂ was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [¹⁸ O]-labeled ¹ O₂ released from thermolabile endoperoxides in association with nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry. The structural characterization by nLC-MS/MS of the amino acids in the tryptic peptides of the proteins showed addition of [¹⁸ O]-labeling atoms in different amino acids.