Leveraging Complementary Ion Activation Methods with Proton Transfer Charge Reduction Reactions for Comprehensive...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Top-down mass spectrometry of large proteins faces challenges with low signal-to-noise ratios (S/N) and spectral congestion.
• Proton transfer charge reduction (PTCR) aids spectral congestion but struggles with low S/N, necessitating extensive spectral averaging.

Purpose of the Study:

• To advance the characterization of large proteins, including monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs).
• To overcome limitations in S/N and spectral congestion in large protein analysis.
• To achieve high sequence coverage and differentiate complex biopharmaceutical structures.

Main Methods:

• Implementation of a hybrid strategy combining ultraviolet photodissociation (UVPD), electron transfer higher collision energy dissociation (EThcD), and PTCR.
• Utilizing liquid chromatography time scale analysis with purposeful chromatographic peak broadening.
• Employing gas-phase fractionation and complementary activation methods.

Main Results:

• Achieved high sequence coverages of 85% for mAb heavy chain (Hc) and 79% for ADC Hc subunits.
• Successfully differentiated two payload positional isomers of an ADC Hc.
• Demonstrated the effectiveness of the hybrid strategy for large protein characterization.

Conclusions:

• The developed hybrid strategy significantly enhances the characterization of large proteins and complex biopharmaceuticals.
• This approach overcomes key limitations in mass spectrometry-based protein analysis.
• Enables precise structural elucidation and isomer differentiation crucial for biopharmaceutical development.