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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Photochemistry

Background:

• Disulfide bonds are crucial for protein structure and function.
• Conventional disulfide bond reduction methods often require harsh conditions incompatible with sensitive biomolecules.
• Mass spectrometry (MS) is a powerful tool for protein analysis, but disulfide bond cleavage can be challenging.

Purpose of the Study:

• To develop a mild, visible light-induced system for disulfide bond reduction.
• To enable compatibility of disulfide bond reduction with peptide and protein sequencing by mass spectrometry (MS).
• To demonstrate the utility of this system in top-down protein MS analysis.

Main Methods:

• Visible light (420 nm) irradiation with thioxanthone (TX) as photocatalyst and 2,3-dihydrofuran (DHF) as cocatalyst.
• Single-electron transfer (SET) mechanism for disulfide bond cleavage.
• Direct detection of reaction intermediates via nanoelectrospray ionization online monitoring and radical trapping.
• Coupling the reduction system with top-down protein MS for sequencing.

Main Results:

• Successful visible light-induced disulfide bond reduction via SET mechanism.
• Direct detection of key radical intermediates (TX radical anion, DHF radical, thiyl radical).
• Achieved 47% sequencing of lysozyme using the coupled system, comparable to conventional methods.
• Demonstrated mild reaction conditions suitable for sensitive biopharmaceuticals.

Conclusions:

• The developed visible light-induced disulfide reduction system is effective and compatible with MS-based protein sequencing.
• This method offers a mild alternative to UV-induced reduction, broadening its applicability in biopharmaceutical analysis.
• The system shows promise for critical applications like disulfide bond mapping in antibodies and antibody-drug conjugates.