Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer - Differential ion mobility–maximizing proteome coverage, selectivity, and sensitivity with FAIMS technology

Significance of the topic

Gas-phase fractionation using FAIMS technology offers an orthogonal separation step in shotgun proteomics workflows. By filtering ions based on differential ion mobility and charge state, FAIMS enhances selectivity and sensitivity, reduces chemical noise, and expands dynamic range. This approach minimizes the need for offline liquid chromatography fractionation, cutting analysis time and resource demands while improving proteome coverage.

Objectives and overview of the study

This study evaluates the performance of the FAIMS Pro interface coupled to the Orbitrap Exploris 240 mass spectrometer. It aims to demonstrate increased proteome depth, reproducible gas-phase fractionation, and high quantitation precision compared to traditional workflows. Key comparisons are made between analyses without FAIMS and those using two compensation voltages to assess peptide identification orthogonality and sensitivity improvements.

Methodology and instrumentation

• Sample preparation: 200 ng HeLa protein digest standard and 10 fmol peptide retention time calibration mixture.
• Liquid chromatography: 25 cm IonOpticks Aurora UHPLC emitter column, 300 nL/min flow rate, 30 min gradient (3 to 95% organic).
• Mass spectrometry: Thermo Scientific EASY-nLC 1200 system, Nanospray Flex ion source, Sonation column oven at 40 °C, FAIMS Pro interface with compensation voltages of -50 V and -70 V, Orbitrap Exploris 240 mass spectrometer operated in data-dependent acquisition mode.
• Data analysis: Proteome Discoverer 2.4 software with 1% peptide spectral match false discovery rate.

Main results and discussion

• Removal of singly charged ions and enrichment of multiply charged peptide precursors enabled by FAIMS Pro, as illustrated by gas-phase spectra comparison.
• Two compensation voltages generated distinct peptide subsets with 90–95% orthogonality, confirmed by Venn diagram analysis of replicate injections.
• Chromatographic traces at each compensation voltage exhibited high reproducibility, supporting consistent peptide identification and quantitation.
• FAIMS-mediated fractionation increased proteome coverage while using minimal sample amounts and a single 30 min gradient.

Benefits and practical applications

By integrating FAIMS Pro into established workflows, laboratories can achieve deeper proteome profiling without additional LC fractionation steps. Enhanced sensitivity and selectivity improve detection of low-abundance peptides, while reproducible gas-phase fractionation streamlines method development and reduces sample consumption. This approach is suited for high-throughput proteomics, quantitative studies, and cases where sample amount or instrument time is limited.

Future trends and opportunities

Advancements in FAIMS hardware and software control are expected to refine compensation voltage stepping and dynamic acquisition strategies. Integration with emerging real-time search algorithms may further enhance identification rates. FAIMS could be paired with novel ion mobility platforms or extended to other omics applications, expanding its utility in multi‐dimensional separation schemes.

Conclusion

The FAIMS Pro interface on the Orbitrap Exploris 240 mass spectrometer delivers robust gas-phase fractionation, increasing proteome depth, selectivity, and quantitative precision. It offers a streamlined, high‐performance alternative to conventional offline fractionation, readily fitting into existing proteomics workflows and enabling comprehensive protein analysis.

References

• Pfammatter P, Bonneil E, McManus FP, et al. Molecular & Cellular Proteomics. 2018 Jul 14.