High-resolution mobility analysis of charge-reduced electrosprayed protein ions

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Biophysics

Background:

• Ion mobility spectrometry (IMS) is valuable for analyzing large biomolecules like protein complexes and viruses.
• Existing IMS methods often yield wide mobility peaks (fwhm ~20%) for electrosprayed ions, limiting resolution.
• This peak broadening is primarily linked to early charge reduction preceding droplet evaporation, which hinders secondary atomization.

Purpose of the Study:

• To investigate and improve the peak width limitations in electrospray ionization (ESI) for IMS analysis of large molecules.
• To develop a method that achieves narrower mobility peaks for singly charged ions.
• To enhance the analytical capabilities of IMS for studying protein complexes and viruses.

Main Methods:

• A novel charge reduction (CR) system was developed, featuring a positively biased electrospray capillary coaxial with a Ni-63 coated chamber generating a bipolar ionic atmosphere.
• A screen was used between the capillary and CR chamber to limit anion penetration, stabilizing the ESI tip and controlling ion recombination.
• The system was tested using ovalbumin and its clusters, with charge-reduced ions analyzed by a differential mobility analyzer (DMA).

Main Results:

• Drying ions before neutralization significantly reduced peak width, achieving a full width at half maximum (fwhm) of ~3.7% for proteins.
• Singly charged ions (z=1) became dominant at ~1 L/min gas flow rates.
• Peak width was influenced by solution impurities, with desalted and acidified solutions yielding the narrowest peaks.
• Removing the screen with sharp nanospray capillaries increased ion transmission but sometimes compromised spray stability.

Conclusions:

• The developed method of drying before neutralizing electrosprayed ions effectively overcomes the limitations of wide mobility peaks in IMS.
• Achieving narrow fwhm (~3.7%) significantly enhances the resolution and analytical power of IMS for large biomolecules.
• The findings pave the way for more precise analysis of protein complexes, viruses, and other large molecular assemblies using ESI-IMS.