Low-resolution FAIMS for increased peptide coverage in low-load and single-cell proteomics

Significance of the topic

Field asymmetric ion mobility spectrometry (FAIMS) is a common front-end filter in low-input and single-cell proteomics to reduce background and improve selectivity. However, standard FAIMS operating modes can trade off sensitivity and coverage when a single compensation voltage (CV) is used. The study demonstrates a practical and instrumentally simple approach—reducing FAIMS resolution by lowering the outer electrode temperature—to increase ion transmission, peptide coverage and quantitative robustness for low-load and single-cell proteomic workflows without additional hardware or major data-processing changes.

Objectives and study overview

The primary aim was to test whether deliberately lowering FAIMS resolution (by decreasing outer electrode temperature from 100°C to 80°C) increases ion transmission and identification numbers in discovery proteomics while keeping the convenience of single-CV acquisition. The study evaluated effects on CV peak shape and transmission using calibration standards, then assessed identification and quantification performance across a HeLa dilution series (250 pg–16 ng) and real single cells (H460), with attention to DIA windowing and injection-time optimization under the altered ion flux.

Methodology and experimental workflow

Key methodological steps and experimental design:

• FAIMS resolution modulation: Outer electrode temperature lowered from 100°C (standard) to 80°C (low-resolution setting) while inner electrode remained 100°C.
• Calibration: FlexMix calibration solution injected to map CV apex, peak full width at half maximum (FWHM) and integrated peak area for several standard m/z values (322.0481, 524.2655, 622.0289, 922.0098).
• Sample series: Thermo Scientific Pierce HeLa peptide digest diluted and injected over a concentration series (250 pg to 16 ng). Single H460 cells isolated, lysed and digested in 384-well One-Pot format using the cellenONE robot.
• Acquisition strategy: Data-independent acquisition (DIA) with one CV (−48 V carrier), DIA window widths and maximum injection times (maxIT) adapted to input load; single cells measured with 20 Th DIA windows and 40 ms maxIT.
• Data analysis: Spectronaut v20.1 used in library-free method-evaluation mode; MS1-level quantification, minimum quantity filter for single-cell data, searches against reviewed human UniProt proteome (UP000005640) plus CRAPome; 1% protein-level FDR.

Used instrumentation

Instrumental configuration used in the study:

• UHPLC: Thermo Neo UHPLC system; trap: PepMap C18 (5 mm × 300 μm i.d.); analytical column: Aurora Rapid 8×75 XT C18 nanoflow column (50°C).
• Mass spectrometer: Thermo Orbitrap Astral mass spectrometer with FAIMS Pro Duo interface and EASY-Spray source.
• MS settings: MS1 in Orbitrap at 240,000 resolution (m/z 400–800), AGC target 500%, maxIT 100 ms. MS2 in Astral (400–800 m/z), AGC 500%; DIA windows and maxIT adjusted per load.
• Sample handling: cellenONE single-cell isolation and on-plate One-Pot lysis/digestion; Hela peptides prepared in 0.1% TFA/0.015% DDM.

Main results and discussion

Findings from calibration and biological samples demonstrate consistent benefits of lowered FAIMS resolution:

• Calibration (FlexMix): Lowering outer electrode temperature broadened CV peaks (increased FWHM) and shifted apex CV slightly toward more positive values, producing larger integrated peak areas (increased ion transmission). For two calibration ions (m/z 524 and 622), transmission at the apex improved by ~18% and ~21%, respectively.
• HeLa 250 pg dilution: Peptide identifications rose substantially when using low-resolution FAIMS—28,063 peptides at standard vs 35,258 at 80°C (+25.6%). Summed protein MS1 quantities and MS1/MS2 fragment ion counts also increased.
• HeLa titration (250 pg–16 ng): Across the series, peptide IDs increased by 14–34% with outer electrode at 80°C. Protein group IDs increased by ~1–10%, with the largest relative gain at 250 pg (e.g., 5,457 vs 4,923 protein IDs, +10% for one parameter set; peptides 39,451 vs 25,895, +34%).
• DIA windowing and injection time: Low-resolution FAIMS yielded higher ion flux, allowing shorter maxIT and enabling narrower DIA windows—this combination increased peptide IDs further. In contrast, narrowing DIA windows under standard FAIMS commonly reduced IDs.
• Single-cell analysis (H460): Lowering FAIMS resolution improved single-cell metrics—protein IDs +4% and peptide IDs +21%—and increased quantified protein quantities. Background (0-cell) controls were also observed; lowering resolution improved sensitivity but introduced differences reminiscent of batch effects (electrode temperature influenced the proteomic fingerprint).
• Precursor characteristics: Overall retention time distribution was unchanged; a slight median m/z shift and a minor increase in +3 charged precursor prevalence were observed with low-resolution FAIMS. Higher-charge precursors (>3+) remained rare.
• Quantitative quality: Increased ion counts at MS1/MS2 under low-resolution FAIMS translated into improved quantitative precision (lower coefficients of variation observed across replicates).

Practical benefits and applications

Practical implications for labs performing low-input and single-cell proteomics:

• Performance improvement is achieved by a simple operational parameter change (outer electrode temperature) without hardware modification or complex acquisition changes.
• Enhanced peptide coverage increases depth of discovery for low-abundance proteins and single cells, improving sensitivity for biological studies where sample is limited.
• Higher ion flux enables method optimization (shorter maxIT, narrower DIA windows) to improve selectivity and throughput simultaneously.
• The approach integrates with common instruments and data analysis pipelines (e.g., Orbitrap/Astral systems and Spectronaut) and is cost-effective.

Future trends and potential uses

Possible directions and broader uses stemming from this work:

• Systematic optimization: Combining electrode-temperature tuning with other FAIMS parameters (CV selection, carrier gas flow) to further tailor ion transmission for specific peptide classes (e.g., high m/z or higher charge states).
• Standardized low-resolution FAIMS protocols for single-cell atlasing to maximize coverage while minimizing duty-cycle penalties from multi-CV strategies.
• Integration with advanced acquisition schemes (adaptive DIA, real-time search triggers) that exploit increased ion flux to improve identification rates and quantitation in ultra-low-input scenarios.
• Investigation of potential batch effects introduced by electrode temperature and development of normalization strategies to maintain comparability across runs.

Conclusions

Lowering FAIMS resolution via a reduction of the outer electrode temperature (from 100°C to 80°C) broadens CV transmission windows, increases ion flux, and substantially expands peptide identification and quantitative robustness in low-load and single-cell proteomics. The effect is most pronounced at very low inputs (e.g., 250 pg), but benefits extend across a broad input range. This operational tweak is simple, cost-effective, and readily implementable on compatible instrumentation, offering an immediate path to improve sensitivity in discovery proteomics without additional hardware or complex data workflows.

References

1. Hoch DG, Belford M, Heil LR, Mechtler K, Matzinger M. Low-resolution FAIMS for increased peptide coverage in low-load and single-cell proteomics. Sci Rep (2026).