In-Source Charge Reduction Enhances Top-Down Characterization of Denatured Proteins

Importance of the Topic

Denatured proteins typically provide enhanced sequence coverage in top-down mass spectrometry, but their broad charge state distributions reduce precursor ion abundance and lead to congested fragmentation spectra. Controlling charge states without compromising structure is crucial to improve signal quality and analytical throughput in structural proteomics.

Objectives and Study Overview

This study aims to assess in-source chemical charge reduction for denatured proteins to achieve native-like charge distributions. By modulating proton transfer in the electrospray source, the approach seeks to improve top-down fragmentation quality and sequence coverage without using nonvolatile buffers.

Methodology

Protein samples (ubiquitin, carbonic anhydrase, bovine serum albumin, Intact Protein Standard Mix) were prepared in denaturing (15% acetonitrile, 0.1% formic acid) and native (100 mM ammonium acetate) conditions. In-source charge reduction was performed by infusing dimethylaminopropylamine via a second nebulizer of a dual-spray electrospray source, adjusting flow rates to control charge states.

Used Instrumentation

• Agilent 1290 Infinity II Bio LC system with PLRP-S column
• Agilent 6545XT AdvanceBio LC/Q-TOF with ExD cell
• Agilent 6560 Ion Mobility LC/Q-TOF with drift tube (nitrogen)

Results and Discussion

• In-source charge reduction successfully narrowed MS1 and MS2 spectra, producing native-like charge states with high ionization efficiency in both direct infusion and denaturing chromatography.
• Top-down ECD of carbonic anhydrase precursors (16+, 22+, 29+) yielded comparable sequence coverage; lower charge states improved signal-to-noise and IonScores. Integrating results from all three charge states increased total coverage to 78%.
• Ion mobility measurements showed charge-reduced precursors retained elongated structures (e.g., CA 20+ CCS ~5973 Å2 vs 5171 Å2 without base; BSA 25+ similarly extended). Extended conformers persisted over time, with larger proteins more stable.
• Comparison to native-like and maximally unfolded structures via CIU indicated that charge-reduced ions adopt conformations akin to collision-induced unfolded states.

Benefits and Practical Applications

In-source charge reduction offers:

• Enhanced control over protein ion charge states without nonvolatile buffers
• Reduced spectral congestion and improved fragmentation quality
• Compatibility with both infusion and chromatographic workflows
• Improved top-down sequence coverage by combining multiple charge states

Future Trends and Potential Applications

This approach could be extended to large proteoforms and protein complexes, integrating with alternative activation methods and high-resolution mobility separations. Automation of charge modulation may enable robust quantification and structural studies in biopharmaceutical characterization and structural proteomics.

Conclusion

In-source chemical charge reduction provides a versatile strategy to refine charge distributions of denatured proteins, yielding cleaner spectra and improved sequence coverage in top-down MS. The retention of extended conformations suggests minimal structural perturbation, supporting its application in advanced proteomic analyses.

References

1. Ugrin SA, English AM, Syka JEP, et al. Ion–Ion Proton Transfer and Parallel Ion Parking for Intact Proteins on a Modified Orbitrap. J Am Soc Mass Spectrom. 2019;30:2163–2173.
2. Lieu LB, Hinkle JD, Syka JEP, Fornelli L. Leveraging Ion–Ion and Ion–Photon Activation to Sequence Proteins with Multiple Disulfide Bonds. J Am Soc Mass Spectrom. 2024;35(12):3265–3273.