Optimization of FAIMS-XL-MS Workflow for Phospho-Enrichable Crosslinkers

Significance of the Topic

Cross-linking mass spectrometry is a powerful technique for probing protein architectures and interaction networks. However, the low yield of cross-linked peptides in complex samples has limited its broader adoption. Integrating high field asymmetric waveform ion mobility spectrometry (FAIMS) introduces an extra gas-phase separation dimension, enhancing sensitivity and selectivity in XL-MS workflows.

Objectives and Overview of the Study

This work benchmarks FAIMS Pro on the Orbitrap Exploris 240 instrument for analysis of phospho-enrichable crosslinkers. Two reagents, DSSO and PhoX-DSPP, were applied to E coli ribosome preparations to optimize FAIMS and MS parameters. The study aims to maximize unique crosslink identifications by adjusting ion mobility settings, ion transfer conditions, gas flows, collision energies, and compensation voltages.

Applied Methodology and Instrumentation

Sample Preparation and Crosslinking

• E coli ribosomal proteins exchanged into PBS pH 7.0 were crosslinked with DSSO or PhoX-DSPP at 30–100× molar excess.
• Reactions were quenched with ammonium bicarbonate, reduced, alkylated, precipitated with acetone, and digested with Lys-C and trypsin.
• PhoX-DSPP digests were enriched via Fe-NTA magnetic beads or agarose prior to LC-MS.

LC-MS Setup

• Chromatography: 75 µm×25 cm EASY Spray column with 75 min gradient at 300 nL/min.
• Mass Spectrometry: Orbitrap Exploris 240 equipped with FAIMS Pro Duo interface.

Data Analysis

• Proteome Discoverer 2.5 with XlinkX node for crosslinks and SEQUEST HT for linear peptides.
• Database search against E coli Uniprot entries at 1% FDR.

Equipment Used

Orbitrap Exploris 240 mass spectrometer equipped with FAIMS Pro Duo interface and Thermo Scientific EASY Spray analytical column.

Main Results and Discussion

Optimization Highlights

• Total carrier gas flow of 0.9 L/min provided stable spray and FAIMS performance.
• Lower spray voltage (1500 V) and transfer tube temperature (250 °C) increased crosslink IDs by approximately 30 compared to standard settings.
• Stepped collision energy at 21 %, 26 % and 31 % was optimal for both DSSO and PhoX-DSPP.

FAIMS Parameter Effects

• DSSO: Two compensation voltages of –45 V and –60 V yielded +40 % unique crosslinks versus no FAIMS.
• PhoX-DSPP enriched samples: CVs of –60 V and –75 V improved identification by 10–14 %.
• Standard resolution mode outperformed high-resolution FAIMS due to greater transmission.

Charge Distribution

• PhoX-DSPP crosslinked peptides predominantly carried 3+ charge, suggesting inclusion of that state improves discovery.

Benefits and Practical Applications

Integrating FAIMS with Orbitrap Exploris 240 XL-MS workflows enhances detection of low-abundance crosslinks and improves enrichment specificity for phospho-tagged reagents. This optimized platform supports detailed mapping of protein interaction networks and structural studies in complex proteomes.

Future Trends and Potential Applications

Upcoming developments may include automated CV scanning, novel enriching crosslinker chemistries, coupling FAIMS with other ion mobility techniques, and applying deep learning to predict optimal FAIMS parameters. Such advances will further expand structural proteomics and drug discovery applications.

Conclusion

Optimizing FAIMS parameters on the Orbitrap Exploris 240 significantly increases unique crosslinked peptide identifications for both cleavable and phospho-enrichable crosslinkers. The refined workflow delivers higher sensitivity and robustness for XL-MS studies of protein complexes.

References

1. Schnirch L, Nadler-Holly M, Siao S, Frese C, Viner R, Liu F. Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry. Analytical Chemistry. 2020;92(15):10495–10503.
2. Steigenberger B, Pieters RJ, Heck A, Scheltema RA. PhoX: An IMAC-Enrichable Cross-Linking Reagent. ACS Central Science. 2019;5(9):1514–1522.
3. Cannon JR, et al. Ultraviolet Photodissociation for Characterization of Whole Proteins on a Chromatographic Time Scale. Analytical Chemistry. 2014;86(4):2185–2192.