Characterizing Macromolecular Dipole Moments via Differential Ion Mobility Spectrometry with Linked Field/Pressure Scans

Significance of the Topic

Macromolecular dipole moments play a key role in defining the structure and behavior of proteins in the gas phase under an electric field. Traditional ion mobility spectrometry (IMS) techniques measure orientationally averaged collision cross sections; however, permanent dipoles contain additional structural information related to secondary and tertiary architecture. By exploiting dipole alignment under strong electric fields, one can gain directional collision cross section values and reveal folding transitions and charge distribution in large biomolecules. This enhances our understanding of protein conformation, supports quality control of biotherapeutics, and advances structural proteomics.

Objectives and Study Overview

The primary goal of the study was to isolate and quantify macromolecular dipole moments using differential ion mobility spectrometry (FAIMS) with linked field and pressure scans. The research aimed to decouple dipole alignment effects governed by absolute electric field strength from other high-field mobility phenomena controlled by the reduced field (E/N). Model proteins albumin (66.5 kDa) and β-galactosidase (105 kDa) were selected to demonstrate the approach on large biomolecules.

Methodology

A modified FAIMS device was integrated into a triple quadrupole mass spectrometer to enable simultaneous variation of dispersion voltage and gas pressure at constant reduced field. Key aspects of the method include:

• Linked scans of dispersion field and nitrogen pressure to maintain constant E/N while tuning absolute electric field strength.
• Infusion of protein standards via electrospray ionization at controlled flow rates into the FAIMS cell.
• Extraction of field asymmetric mobility spectra to resolve aligned and rotationally averaged ion populations.

Instrumentation

Experimental work was conducted on a Shimadzu LCMS-8060 system modified to include a vacuum differential mobility spectrometry (vDMS) cell positioned between the curtain gas inlet and the first quadrupole. Features of the setup:

• Electrospray ionization source infusing samples at 9 μL/min.
• vDMS cell operated with nitrogen at pressures ranging from 35 to 245 mbar, controlled by pumping and gas addition.
• Rectangular waveform at 200 kHz frequency and 2.5 aspect ratio to generate the dispersion field.
• Detection via a triple quadrupole configuration enabling mass selection of specific charge states.

Main Results and Discussion

Key findings of the pressure‐dependent FAIMS experiments include:

• At low pressures, mobility spectra display single Gaussian peaks corresponding to rotationally averaged conformers.
• As pressure increases, low‐field tails emerge and grow into distinct peaks attributed to dipole‐aligned ions.
• The threshold pressure for alignment decreases for higher charge states and larger proteins, consistent with theory predicting alignment for dipoles above approximately 300 Debye at ambient conditions.
• Dipole moment histograms derived from the fraction of aligned conformers reveal unfolding transitions in albumin around charge state z≈40–50.
• Directional collision cross sections for aligned states are lower than averaged values, reflecting orientation along the molecular long axis.

Benefits and Practical Applications

This method provides a direct way to quantify permanent dipole moments and directional collision cross sections of large biomolecules. Practical advantages include enhanced conformational characterization in proteomics, improved structural validation for biopharmaceutical quality control, and new analytical dimensions for studying protein folding, complex assembly, and charge distribution.

Future Trends and Opportunities

Future developments may involve integrating high‐resolution IMS stages before and after FAIMS for tandem mobility analyses, applying the approach to non‐protein macromolecules and complexes, and coupling real‐time pressure control with advanced waveform designs to probe dynamic conformational changes under field‐induced alignment.

Conclusion

The study demonstrates that linked field/pressure scans in FAIMS enable robust extraction of macromolecular dipole moments and directional collision cross sections. By tuning gas pressure at constant E/N, dipole alignment can be selectively controlled, offering a powerful tool for detailed structural analysis of large biomolecules in the gas phase.

References

1. Shvartsburg AA, Bryskiewiecz T, Purves RW, Tang K, Guevremont R, Smith RD. Field Asymmetric Waveform Ion Mobility Spectrometry Studies of Proteins: Dipole Alignment in Ion Mobility Spectrometry? J Phys Chem B. 2006;110:21966.
2. Shvartsburg AA, Noskov SY, Purves RW, Smith RD. Pendular Proteins in Gases and New Avenues for Characterization of Macromolecules by Ion Mobility Spectrometry. Proc Natl Acad Sci USA. 2009;106:6495.
3. Shvartsburg AA, Andrzejewski R, Entwistle A, Giles R. Ion Mobility Spectrometry of Macromolecules with Dipole Alignment Switchable by Varying the Gas Pressure. Anal Chem. 2019;91:8176.