Acetone/Isopropanol Photoinitiating System Enables Tunable Disulfide Reduction and Disulfide Mapping via Tandem Mass Spectrometry.

Herein, we report the development of a new photochemical system which enables rapid and tunable disulfide bond reduction and its application in disulfide mapping via online coupling with mass spectrometry (MS). Acetone, a clean and electrospray ionization (ESI) compatible solvent, is used as the photoinitiator (1% volume) in the solvent system consisting of 1:1 alkyl alcohol and water. Under ultraviolet (UV) irradiation (∼254 nm), the acetone/alcohol system produces hydroxyalkyl radicals, which are responsible for disulfide bond cleavage in peptides. Acetone/isopropanol is most suitable for optimizing the disulfide reduction products, leading to almost complete conversion in less than 5 s when the reaction is conducted in a flow microreactor. The flow microreactor device not only facilitates direct coupling with ESI-MS but also allows fine-tuning of the extent of disulfide reduction by varying the UV exposure time. Near full sequence coverage for peptides consisting of intra- or interchain disulfide bonds has been achieved from complete disulfide reduction and online tandem mass spectrometry (MS/MS) via low energy collision-induced dissociation. Coupling different degrees of partial disulfide reduction with ESI-MS/MS allows disulfide mapping as demonstrated for characterizing the three disulfide bonds in insulin.