Mapping Complex Disulfide Bonds via Implementing Photochemical Reduction Online with Liquid Chromatography–Mass Spectrometry

Assigning disulfide linkage is a crucial task for protein identification. The current bottom-up proteomics workflow has limitations in characterizing peptide digests containing multiple disulfide bonds due to the difficulty of controlling partial reduction via conventional chemical reduction methods. Previously, our lab reported the development of an acetone/2-propanol (IPA) photoinitiating system for rapid (on second time scale) and tunable disulfide bond reduction. Herein, we incorporated this reaction system onto a liquid chromatography-mass spectrometry (LC-MS) system for bottom-up protein analysis applications. The photochemical reduction reaction was implemented in a flow microreactor which allowed for up to 15 s 254 nm UV irradiation. The microreactor was installed post LC separation and right before electrospray ionization, while a T-junction was used to introduce the photoinitiating solution to the LC eluent before entering the microreactor. The degree of disulfide reduction was tunable from partial reduction to complete reduction for peptides containing one or multiple disulfide bonds. Significantly improved sequence coverage was obtained from complete disulfide reduction, while assignment of the disulfide connectivity was facilitated from partial disulfide reduction when coupled with tandem mass spectrometry via collision-induced dissociation. As a proof-of-concept test, trypsin digests of lysozyme (four disulfide bonds) and bovine serum albumin (BSA, 17 disulfide bonds) were analyzed by the LC-MS system coupled with online reduction. Sequence coverage was improved from 35% to 100% and 13% to 87% for lysozyme and BSA, respectively. All four disulfide bonds of lysozyme were determined. For BSA, nine disulfide bonds were characterized and eight adjacent disulfide bonds were narrowed down.