High-Coverage Disulfide Mapping Enabled by Programmable Disulfide-Ene Reaction Integrated onto a Bottom-Up Protein Analysis Workflow

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