Ultrahigh-resolution differential ion mobility spectrometry using extended separation times.

Area of Science:

• Analytical Chemistry
• Separation Science
• Biomolecular Analysis

Background:

• Field asymmetric waveform ion mobility spectrometry (FAIMS) is a powerful tool for gas-phase ion separation, often coupled with mass spectrometry.
• Historically, FAIMS resolution (R) was limited to approximately 20, hindering detailed analysis.
• Previous advancements using stronger fields and optimized gases reached R ~200 but faced electrical breakdown limitations.

Purpose of the Study:

• To enhance the resolving power of planar FAIMS devices beyond current limitations.
• To improve the separation and identification of complex peptide mixtures, including isomers.
• To extend the applicability of FAIMS in proteomic and metabolomic analyses.

Main Methods:

• Extended the separation time (t) in planar FAIMS devices by reducing carrier gas flow, up to fourfold.
• Utilized the established relationship between resolving power and separation time (R ∝ √t).
• Applied the enhanced FAIMS technique to analyze tryptic digests of peptides.

Main Results:

• Achieved a significant increase in FAIMS resolving power, reaching over 300 for multiply charged peptides.
• Demonstrated the separation of previously unresolved peptide isomers, such as folding conformers and modified peptide variants.
• Reached a peak capacity of approximately 200 in the analysis of tryptic digests.

Conclusions:

• Extended separation times in FAIMS offer a viable route to substantially increase resolving power without electrical breakdown.
• The improved resolution enables the differentiation of complex peptide isomers, advancing biomolecular analysis.
• This work significantly broadens the utility of FAIMS for high-resolution separations in proteomics and related fields.