From Congestion to Clarity: On the Complementarity of Resolving Power and Spectral Simplification for Intact Protein...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down mass spectrometry (TDMS) is crucial for analyzing intact proteins and post-translational modifications (PTMs).
• Challenges exist in interpreting complex spectra for proteins >30 kDa, even with advanced fragmentation techniques and high mass resolution.
• Spectral congestion hinders accurate analysis of large proteins.

Purpose of the Study:

• To evaluate the impact of increased resolving power and proton transfer charge reduction (PTCR) on TDMS.
• To assess spectral simplification and sequence coverage for large intact proteins.
• To apply the optimized TDMS approach to biopharmaceutical characterization.

Main Methods:

• Utilized Tribrid Orbitrap mass spectrometry with alternative fragmentation techniques.
• Compared TDMS performance at low and high mass resolving powers (60,000 vs. 480,000 at m/z 200).
• Applied proton transfer charge reduction (PTCR) for spectral simplification.
• Analyzed enolase, carbonic anhydrase, myoglobin, and ubiquitin, as well as NIST monoclonal antibody subunits.

Main Results:

• High resolving power increased carbonic anhydrase sequence coverage from 50.5% to 92.6%.
• PTCR further enhanced coverage to 97.7% for carbonic anhydrase.
• The combined approach successfully characterized digested and reduced NIST mAb subunits.

Conclusions:

• Increasing resolving power and employing PTCR are effective strategies to overcome spectral complexity in TDMS.
• This optimized TDMS method enhances proteoform characterization and PTM localization for large proteins.
• The approach is valuable for the structural analysis of complex biopharmaceuticals.