Singlet oxygen‐induced protein aggregation: Lysozyme crosslink formation and nLC‐MS/MS characterization

Singlet molecular oxygen (¹O₂) has been associated with a number of physiological processes. Despite the recognized importance of ¹O₂‐mediated protein modifications, little is known about the role of this oxidant in crosslink formation and protein aggregation. Thus, using lysozyme as a model, the present study sought to investigate the involvement of ¹O₂ in crosslink formation. Lysozyme was photochemically oxidized in the presence of rose bengal or chemically oxidized using [¹⁸O]‐labeled ¹O₂ released from thermolabile endoperoxides. It was concluded that both ¹O₂ generating systems induce lysozyme crosslinking and aggregation. Using SDS‐PAGE and nano‐scale liquid chromatography coupled to electrospray ionization mass spectrometry, the results clearly demonstrated that ¹O₂ is directly involved in the formation of covalent crosslinks involving the amino acids histidine, lysine, and tryptophan.