High-Coverage\nDisulfide Mapping Enabled by Programmable\nDisulfide-Ene Reaction Integrated onto a Bottom-Up Protein Analysis\nWorkflow

Mapping disulfide linkages is crucial for characterizing\npharmaceutical\nproteins during drug development and quality control. Traditional\nbottom-up protein analysis workflows often suffer from incomplete\nmapping for tryptic peptides consisting of multiple disulfide bonds.\nAlthough the employment of a partial reduction of disulfide bonds\ncan improve disulfide mapping, it becomes a bottleneck of analysis\nbecause individual tuning is often needed. Herein, we have developed\nan online disulfide-ene reaction system in which the composition of\nthe reaction solvent can be programmed to achieve optimal partial\nreduction of tryptic disulfide peptides after liquid chromatography\nseparation. By coupling this system onto a bottom-up protein analysis\nworkflow, high coverage for sequencing (71–83%) and disulfide\nmapping (84–100%) was achieved for standard proteins consisting\nof 4–19 disulfide bonds. The analytical capability was further\ndemonstrated by mapping 13 scrambled disulfide bonds in lysozyme and\nachieving compositional analysis of IgG isotypes (κ and λ)\nand subclasses (IgG1–IgG4) from human plasma.