High-Pressure Electrospray Ionization Yields Supercharged Protein Complexes from Native Solutions While Preserving...

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Structural Biology

Background:

• Native mass spectrometry (MS) aims to study protein complexes in the gas phase, preserving noncovalent interactions.
• Increasing protein complex charge states (supercharging) is crucial for deeper structural insights but can affect native structures.
• The capability of high-pressure supercharging for large protein assemblies and its structural impact remain largely unexplored.

Purpose of the Study:

• To investigate the efficacy of high-pressure-induced supercharging for large protein complexes under native conditions.
• To determine how high-pressure supercharging affects the gas-phase structures and topologies of protein assemblies.
• To explore the relationship between protein properties (MW, surface area) and supercharging efficiency.

Main Methods:

• Applied a high-pressure-induced supercharging strategy in the ion source of a mass spectrometer.
• Analyzed 32 proteins and protein complexes (8.58–801 kDa) using native MS.
• Measured collision cross section (CCS) variations as a function of charge state to probe gas-phase structures.

Main Results:

• Achieved supercharging of protein complexes beyond the Rayleigh limit (Z_R) while preserving native-like topology.
• Demonstrated that increased average charge states strongly correlate with protein surface area and molecular weight (MW).
• Observed that smaller proteins exhibit larger CCS variations upon supercharging, while large complexes are less affected.
• Identified surface charge density and charged surface basic residues as key factors influencing CCS-charge relationships.

Conclusions:

• High-pressure supercharging is a rapid, simple, and controlled method for increasing protein ion charge states in native MS.
• This technique effectively supercharges large protein assemblies beyond Z_R without compromising native topology.
• The findings facilitate advanced native top-down MS analyses, improving ion transmission, fragmentation, and detection efficiency.