Reliable and Deep Proteome Coverage by Gas-Phase Fractionation of Peptides with a FAIMS Pro Interface on a Modified Quadrupole Orbitrap

Significance of the Topic

Proteome analysis of complex biological samples remains challenging due to high dynamic range and sample complexity. Gas-phase fractionation using FAIMS Pro™ offers an inline, rapid alternative to time-consuming offline pre-fractionation methods, improving sensitivity for low-abundance peptides and reducing chemical noise.

Study Objectives and Overview

The study aimed to evaluate and optimize a method that integrates the Thermo Scientific™ FAIMS Pro interface with the Orbitrap Exploris™ 480 mass spectrometer. By comparing single compensation voltage (CV) runs and CV-switching experiments under both nano-flow and capillary-flow LC conditions, the authors sought to maximize protein identifications while preserving peptide coverage.

Methodology and Instrumentation

A commercially available tryptic HeLa digest standard (500 ng/μL) was analyzed in data-dependent acquisition mode. Three gradient lengths (30, 60, 90 min) and two flow regimes (nano-flow at 300 nL/min, capillary flow at 1,000 nL/min) were tested. MS1 and MS2 parameters were adjusted for optimal AGC targets and resolution. Peptide identification employed Proteome Discoverer™ 2.4 with SEQUEST® HT, using fixed carbamidomethylation and variable oxidation, deamidation, and acetylation modifications. False discovery rate was set at 1% for peptides and proteins.

Instrumentation Details

• Mass spectrometer: Orbitrap Exploris™ 480 equipped with FAIMS Pro™ interface
• LC systems: EASY-nLC™ 1200 with PepMap™ C18 columns (75 µm×50 cm for nano-flow; 150 µm×15 cm for capillary flow)
• Software: Thermo Scientific™ Proteome Discoverer™ 2.4, SEQUEST® HT search engine

Main Results and Discussion

Single-CV FAIMS runs reduced peptide identifications relative to no-FAIMS baselines but increased protein group identifications by up to 15%. Implementing two-CV or three-CV switching within a single LC-MS run restored peptide coverage and further boosted protein IDs without extending analysis time. Specific observations:

• CV = ‑40 V favored high-mass precursors; CV = ‑65 V enriched low-mass precursors.
• Overlap of peptide identifications between single CVs was <10%, and between three CVs <4%, indicating complementary gas-phase fractions.
• Under capillary-flow 30 min gradients, single-CV experiments achieved up to 25% more protein IDs versus no-FAIMS, with no peptide loss.

Practical Benefits and Applications

FAIMS Pro integration enables deeper proteome coverage with minimal sample loss and no additional offline fractionation steps. The method is compatible with both nano-flow and higher throughput capillary-flow workflows, making it suitable for laboratories focused on high-sensitivity discovery proteomics, QA/QC, and time-sensitive industrial analytics.

Future Trends and Opportunities

Further developments may include adaptive CV-selection algorithms driven by machine learning, integration of FAIMS with multiplexed quantitative strategies, and combination with emerging ion-mobility technologies. Optimization for targeted workflows and high-throughput screening could expand FAIMS application domains.

Conclusion

The FAIMS Pro interface on the Orbitrap Exploris™ 480 significantly enhances protein identification rates across various gradient lengths and flow regimes. CV switching offers a rapid, robust gas-phase fractionation strategy that complements existing proteomics workflows without increasing sample preparation or instrument run time.

References

1. A. Hebert et al., Anal. Chem., 2018, 90, 9529–9537.
2. Pfammatter et al., Mol. Cell Proteomics, 2018, 17(10), 2051–2067.