Deep Proteomic Coverage Using Fast and Sensitive FAIMSDevice Coupled to a Thermo Scientific Orbitrap Fusion Lumos Tribid Mass Spectrometer

Significance of the Topic

The combination of high-field asymmetric waveform ion mobility spectrometry (FAIMS) with Orbitrap Fusion Lumos mass spectrometers significantly enhances proteomic depth by reducing chemical background and enriching multiply charged ions, thereby improving sensitivity and throughput in bottom-up analyses.

Objectives and Study Overview

This study evaluated the impact of FAIMS ion transit time on mass spectrometer performance and optimized compensation voltage (CV) switching strategies for deep proteome coverage during a 1.5 hour LC gradient of a HeLa peptide digest.

Methodology and Instrumentation

Analysis was performed on Thermo Scientific Orbitrap Fusion and Orbitrap Fusion Lumos instruments equipped with a FAIMS Pro interface. A 1 µg HeLa digest was separated on a 75 µm×50 cm C18 column using a 1.5 hour gradient. CVs from –30 V to –120 V were screened to identify optimal settings. Two FAIMS ion transit times (40 ms and 0.1 ms) and multiple CV switching schemes (2 to 5 CVs with turbo switching) were compared. MS parameters included HCD at 60 000 resolution, AGC targets, and ion injection times from 10 ms to 35 ms. Parallel MS1–MS2 acquisition with APD enabled high scan rates. Data processing was carried out with Proteome Discoverer 2.1.

Results and Discussion

• Reducing FAIMS ion transit time from 40 ms to 0.1 ms eliminated duty-cycle overhead, enabling four CVs (–48, –55, –65, –72 V) without performance penalty and yielding over 53 000 unique peptides and 6600 proteins in a single run.
• FAIMS attenuated precursor signal roughly two-fold but significantly improved MS1 precursor purity and reduced chemical background, leading to ~40 % more peptide identifications compared to no FAIMS.
• Optimal CVs (–50 V and –70 V) enriched for 2+ and 3+ charge states, maximizing peptide-spectrum matches (PSM), peptide, and protein identifications.
• Turbo CV switching with four CVs achieved the highest identification rates, with MS2 acquisition up to 35 Hz and peptide ID rates above 12 peptides per second.
• Dynamic CV scheduling (CV-RT) further increased peptide identifications to over 51 700 with minimal change in protein coverage.
• Ion injection times optimized at 20 ms provided a balance between scan speed and identification performance without impacting protein yield.

Benefits and Practical Applications

• Enhanced detection of low-abundance peptides via gas-phase enrichment.
• High-throughput deep proteome profiling with negligible duty-cycle penalties.
• Suitable for quantitative bottom-up proteomics, quality control workflows, and analysis of complex biological samples.

Future Trends and Potential Applications

• Implementing FAIMS at near-100 % duty cycle for routine proteomic workflows.
• Development of advanced real-time CV selection and adaptive switching algorithms.
• Extension of FAIMS integration with data-independent acquisition and post-translational modification mapping.
• Translation into clinical proteomics and targeted biomarker discovery studies.

Conclusion

Shortening FAIMS ion transit time to 0.1 ms and employing turbo CV switching strategies enable comprehensive proteome coverage without acquisition penalties. FAIMS delivers substantial improvements in precursor purity, identification rates, and sensitivity, establishing a robust platform for high-throughput bottom-up proteomics.

Used Instrumentation

• Thermo Scientific Orbitrap Fusion and Orbitrap Fusion Lumos Tribrid mass spectrometers
• FAIMS Pro interface with RF generator and electrode assembly
• Thermo Scientific EASY-nLC 1000 system with EASY-Spray 75 µm×50 cm C18 column
• APD cell for parallel MS1–MS2 acquisition