Evaluation of FAIMS Technology for Mass Spec Analysis of Chemical Cross-linked Peptides

Significance of the Topic

Chemical cross-linking combined with mass spectrometry is a critical approach for mapping protein-protein interactions and structural architectures. However, the low abundance of cross-linked peptides and complexity of biological samples limit identification rates. High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) introduces an orthogonal, gas-phase separation that enhances dynamic range, reduces chemical noise and improves quantitation accuracy, making it a promising tool to boost cross-linking mass spectrometry workflows.

Study Objectives and Overview

This study aimed to evaluate the performance of a FAIMS Pro device on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer for analysis of chemical cross-linked peptides. Enrichment and fractionation methods (strong cation exchange, size exclusion chromatography) were compared against gas-phase FAIMS separation, both alone and in combination, across simple protein standards and complex biological samples including bovine serum albumin (BSA), yeast enolase, HeLa cell lysate and mouse heart mitochondria.

Methodology and Instrumentation

Sample Preparation and Fractionation:

• Crosslinkers: DSS, DSSO, DSBU applied to BSA, enolase, cell lysates and mitochondria.
• Digestion: Reduction, alkylation, trypsin proteolysis; SCX spin columns or SEC for offline fractionation.

Mass Spectrometry and FAIMS:

• LC Systems: Dionex UltiMate 3000 and EASY-nLC 1200 UPLC with C18 columns and 45–120 min gradients.
• MS Platform: Orbitrap Fusion Lumos Tribrid with Thermo FAIMS Pro interface.
• FAIMS Settings: Compensation voltages evaluated between –40 V and –85 V; internal stepping (intra-analysis CV cycling) and external stepping (inter-analysis CV switching).

Data Analysis:

• Software: Proteome Discoverer 2.3 with XlinkX 2.0 node for cross-links and SEQUEST HT for unmodified peptides.
• Search Parameters: 1% FDR, specific cross-link modification masses, XlinkX score ≥40, ΔXlinkX ≥4.

Key Results and Discussion

• Internal FAIMS CV stepping with three voltages consistently outperformed single CV and external stepping, yielding up to two-fold more cross-linked spectral matches.
• Combination of FAIMS with SCX or SEC fractionation provided similar or better identification numbers compared to traditional multi-fraction workflows, while reducing offline steps.
• In complex mitochondrial samples, two-dimensional FAIMS-SCX analysis doubled the number of identified inter- and intra-protein contacts, increasing separation peak capacity.
• FAIMS reduced interference in tandem-mass-tag (TMT) labeled cross-link quantitation, enhancing both identification and quantitative precision.

Benefits and Practical Applications

By integrating FAIMS into cross-linking MS pipelines, laboratories can achieve higher cross-link identification rates in fewer runs, reduce sample complexity, and improve quantitation accuracy for multiplexed experiments. This approach streamlines workflows, minimizes instrument time and enhances detection of low-abundance interactions in proteome-wide studies.

Future Trends and Potential Applications

Future developments may include real-time adaptive CV selection, integration with additional ion mobility technologies, longer LC gradients for deeper coverage and applications to intact organelles or in vivo cross-linking workflows. Expansion of FAIMS CV libraries for diverse crosslinkers and machine-learning-driven CV optimization could further boost sensitivity and throughput.

Conclusion

This work demonstrates that FAIMS Pro on an Orbitrap Fusion Lumos significantly enhances the analysis of chemical cross-linked peptides, both as a standalone separation and in tandem with SCX/SEC fractionation. Adoption of optimized multi-CV FAIMS methods offers a robust path to improved protein interaction mapping in complex biological systems.

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