Comprehensive Evaluation of Bottom-up Proteomics using an Orbitrap Fusion Lumos Tribrid Mass Spectrometer with FAIMS Pro Interface

Significance of the Topic

Bottom-up proteomics is a cornerstone technique for identifying and quantifying proteins in complex biological samples. The high dynamic range and molecular complexity of proteomes often limit protein coverage and quantification accuracy. Implementing gas-phase ion mobility separation prior to mass analysis addresses these challenges by reducing spectral interferences and enhancing sensitivity.

Objectives and Study Overview

This work evaluates the performance of the Thermo Scientific FAIMS Pro Interface coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Key applications studied include single-shot proteome profiling, phosphoproteomics, reporter-ion quantification (TMT11plex), and crosslinking-based structural biology. The goal is to assess gains in depth, precision, and structural insight achieved through FAIMS-enabled gas-phase fractionation.

Methodology and Used Instrumentation

Samples analyzed comprised tryptic digests of human cell lines (HeLa, 293T), depleted plasma, enriched phosphopeptides, and DSS-crosslinked BSA. Liquid chromatography was performed on an Easy-nLC 1200 system with nano-flow columns. The FAIMS Pro interface was operated at multiple compensation voltages to selectively transmit ion populations. Data-dependent acquisition employed a top-speed cycle with Orbitrap full MS scans and ion-trap HCD MS/MS scans. Data processing used Proteome Discoverer 2.3 with XlinkX2.0 for crosslink analysis.

• Orbitrap Fusion Lumos Tribrid Mass Spectrometer
• FAIMS Pro Interface with adjustable Dispersion and Compensation Voltages
• Thermo Scientific Easy-nLC 1200 nano-LC system
• Proteome Discoverer software v2.3 with XlinkX2.0

Key Results and Discussion

• FAIMS increased peptide and protein identifications by 10–20% across single-shot HeLa and 293T digests compared to analyses without FAIMS.
• At 200 ng HeLa digest, ~5 000 proteins per hour were identified (12% improvement).
• Using 1 µg 293T digest, ~7 000 proteins at 1% FDR were identified in a single 2 h run (24% more peptides, 13% more proteins).
• In TMT11plex yeast standards, FAIMS reduced precursor interference: over 90% of channels showed <50% isolation interference, enabling high-precision quantification via SPS-MS3.
• Depleted plasma and phosphopeptide samples yielded >700 proteins and ~7 000 peptides in a one-hour gradient, demonstrating improved dynamic range coverage.
• DSS crosslinking of BSA identified nearly double the crosslinks and spectrum matches, enhancing structural detail in protein interaction analyses.

Benefits and Practical Applications

The FAIMS Pro interface provides a robust gas-phase fractionation step that boosts proteome depth and quantification precision. It is especially valuable for samples with wide dynamic ranges (e.g., body fluids, PTM-enriched fractions) and for structural proteomics workflows requiring high-charge species discrimination.

Future Trends and Applications

Integrating FAIMS with data-independent acquisition (DIA) strategies, chromatographic and spectral libraries will further enhance single-shot whole-proteome coverage. Emerging directions include comprehensive PTM mapping, targeted low-abundance biomarker quantification, and refined crosslinking analyses for interactome and structural studies.

Conclusion

Coupling the FAIMS Pro interface to an Orbitrap Fusion Lumos Tribrid mass spectrometer significantly enhances sensitivity, selectivity, and throughput in bottom-up proteomics. The approach delivers deeper proteome coverage, superior quantification accuracy, and richer structural insights, facilitating novel discoveries in complex biological systems.

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