Visible-Light-Induced Disulfide Bond Reduction in Peptides and Proteins

Herein, we report a visible light-induced disulfide bond reduction system compatible with peptide and protein sequencing by mass spectrometry (MS). The reaction proceeds via a single-electron transfer (SET) mechanism, utilizing thioxanthone (TX) as the photocatalyst and 2,3-dihydrofuran (DHF) as the cocatalyst. During the process, TX is first exited to its triplet state under 420 nm light irradiation, followed by SET from DHF to generate a TX radical anion. Disulfide bond cleavage occurs promptly after SET from the TX radical anion, yielding the thiol products. Major intermediates, including the TX radical anion, DHF radical, and thiyl radical, have been directly detected by nanoelectrospray ionization online monitoring or after radical trapping. As a proof-of-concept, we have coupled the visible light-induced disulfide reduction with top-down protein MS analysis, achieving 47% sequencing for lysozyme (4 disulfide bonds, 14 kDa), with performance comparable to conventional methods. In contrast to UV-induced disulfide reduction systems, the mild conditions of the visible light-induced reduction make it promising for further development in the analysis of biopharmaceuticals, such as antibodies and antibody-drug conjugates, where sequencing and disulfide bond mapping are critical.