Evaluation of Novel FAIMS Technology for Intact Protein Detection and Characterization by Infusion

Importance of the Topic

Intact protein analysis by mass spectrometry is a critical tool in proteomics, enabling direct detection of proteoforms without digestion. Complex biological samples, such as cell lysates, pose challenges due to wide dynamic ranges, charge state distributions, and singly charged species that can dominate the signal. FAIMS (high‐Field Asymmetric waveform Ion Mobility Spectrometry) offers gas‐phase separation of ions by mobility, potentially enhancing signal clarity and proteoform detection in infusion‐based workflows.

Objectives and Study Overview

The study evaluated a novel cylindrical FAIMS interface coupled to a Thermo Scientific™ Orbitrap™ Fusion™ Lumos™ Tribrid™ mass spectrometer. Goals included optimizing FAIMS settings for simple protein mixtures, comparing intact protein detection with and without FAIMS, and assessing whether FAIMS could reduce or eliminate solid‐phase extraction (SPE) cleanup in E. coli lysate analysis.

Methodology and Instrumentation

The workflow involved:

• Preparation of standard mixtures (seven‐protein “Lucky 7” mix) and E. coli lysates, both pre‐ and post‐SPE cleanup.
• Infusion of samples at nanoflow rates using a syringe pump with EASY‐Spray™ emitter or TriVersa NanoMate™.
• FAIMS parameters: dispersion voltage (DV) –5000 V, compensation voltage (CV) stepped from –100 V to +60 V in 10–20 V increments, electrode temperature at 100 °C.
• Mass spectrometry settings: MS1 resolutions of 7.5, 30, 60, and 120 k, AGC target 2 × 10^6, maximum injection time 100 ms, 5–10 microscans.
• Data processing with Thermo FreeStyle™ software for deconvolution and an in‐house UI for proteoform counting and gel‐plot visualization.

Key Results and Discussion

FAIMS separation markedly improved the number of detected proteoforms in both simple and complex samples. In the Lucky 7 mix, optimal CV ranges (–80 V to +60 V) were identified for each protein, with several species only observed when FAIMS was enabled. In E. coli lysates, FAIMS enhanced detection compared to no FAIMS, and similar proteoforms were identified in both non‐desalted and SPE‐cleaned samples under FAIMS. Non‐desalted lysates showed higher 1+ ion populations without FAIMS, whereas FAIMS mitigated this issue, facilitating detection of multiply charged proteoforms. SPE cleanup still yielded the highest proteoform count, especially at more positive CVs.

Benefits and Practical Applications

• Increased proteoform coverage in infusion‐based experiments without chromatographic separation.
• Gas‐phase filtering reduces interference from singly charged species and simplifies sample preparation.
• Potential to bypass or minimize SPE cleanup steps for rapid screening of protein mixtures or cell lysates.

Future Trends and Potential Applications

Future developments may include integration of FAIMS with high‐throughput workflows, automated CV optimization for diverse sample types, and coupling with top‐down fragmentation strategies for improved proteoform characterization. Emerging ion mobility technologies and machine‐learning algorithms may further enhance selectivity and throughput in intact protein analysis.

Conclusion

The cylindrical FAIMS interface significantly enhances intact protein detection in infusion‐based MS by selectively transmitting multiply charged ions and filtering unwanted species. While SPE cleanup remains beneficial, FAIMS can reduce sample preparation complexity and deliver robust proteoform profiling in simple and complex mixtures.

Reference

• Purves RW; Guevremont R. Anal. Chem. 1999, 71, 2346–2357.