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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Disulfide bond mapping is vital for pharmaceutical protein characterization and quality control.
• Traditional methods struggle with incomplete mapping of complex tryptic peptides.
• Partial disulfide bond reduction is effective but often requires time-consuming individual tuning.

Purpose of the Study:

• To develop an automated system for optimal partial reduction of disulfide bonds in tryptic peptides.
• To improve the efficiency and coverage of disulfide bond mapping in bottom-up proteomics.
• To enhance the characterization of pharmaceutical proteins and complex biological samples.

Main Methods:

• Development of an online disulfide-ene reaction system coupled with liquid chromatography.
• Programming reaction solvent composition for optimal partial reduction of disulfide bonds.
• Application of the system to a bottom-up protein analysis workflow.

Main Results:

• Achieved high sequencing coverage (71-83%) and disulfide mapping coverage (84-100%) for proteins with 4-19 disulfide bonds.
• Successfully mapped 13 scrambled disulfide bonds in lysozyme.
• Enabled compositional analysis of IgG isotypes and subclasses from human plasma.

Conclusions:

• The online disulfide-ene reaction system significantly improves disulfide mapping efficiency and coverage.
• This method offers a robust solution for analyzing complex protein structures in drug development and quality control.
• The system demonstrates broad applicability in characterizing protein modifications and complex biological mixtures.