Extended lifespan of innovative column assemblies in low-flow ion sources

Significance of the Topic

Modern proteomics experiments frequently involve hundreds to thousands of samples and demand stable, reproducible low-flow LC-MS performance over extended injection series. Column clogging, emitter degradation and backpressure rise represent critical challenges that limit throughput and data consistency. Innovative cartridge assemblies with integrated emitters and automated positioning can address these issues and enable large-scale proteome profiling with minimal downtime.

Aims and Study Overview

This work evaluates longevity and reproducibility of two novel ion source cartridges – the OptiSpray µPAC Neo HT and the PepMap Neo 75 µm × 15 cm with FAIMS – across thousands of injections of HeLa and human plasma digests. Key objectives are to quantify protein and peptide identification stability, chromatographic precision and the impact of emitter replacement and cartridge removal and reinstallation.

Methodology and Instrumentation

Sample workflows

• HeLa digest: Thermo Scientific Pierce HeLa Protein Digest reconstituted in 0.1 % FA or TFA, spiked with PRTC standard
• Plasma digest: Human blood plasma processed via S-Trap mini spin column digestion

LC-MS platforms

• Vanquish Neo UHPLC coupled to Orbitrap Exploris 480 or Exploris 240 mass spectrometers
• FAIMS Pro Duo interface applied on the PepMap Neo cartridge runs
• TSQ Quantis and TSQ Altis instruments used in aging experiments for cartridge transfers

Data acquisition and analysis

• Data-dependent acquisition (DDA) for most HeLa digest studies
• Data-independent acquisition (DIA) for plasma digest on Exploris 240
• Proteome Discoverer with CHIMERYS and INFERYS, Spectronaut directDIA and Skyline for PRTC quantitation

Main Results and Discussion

The µPAC Neo HT cartridge delivered over 2000 HeLa injections with protein and peptide identification CVs below 2.1 % and 2.0 %, respectively. Identification counts decreased by less than 10 % after 2000 runs and remained within 9 % even after emitter replacement at 1000 injections. Retention time rolling CVs were consistently below 0.5 %.
On the PepMap Neo 75 µm × 15 cm cartridge with FAIMS, 500 HeLa injections achieved peak area CVs under 11 % and RT CVs under 2.8 %, with median RT stability at 0.1 %.
Plasma digest longevity tests on the µPAC Neo HT cartridge across 211 injections demonstrated protein and peptide ID CVs under 0.6 % and 0.5 %, as well as median peak width and RT deviations below 1.5 seconds.
Automated emitter positioning and cartridge reinstallations introduced negligible variability, highlighting the robustness of the OptiSpray source platform.

Benefits and Practical Applications

• High-throughput compatibility with minimal maintenance over thousands of injections
• Low run-to-run and cartridge-to-cartridge variability for both complex HeLa and plasma digests
• Integrated emitter replacement extends cartridge life without compromising data quality
• Automated positioning reduces manual intervention and enhances reproducibility

Future Trends and Opportunities

Further development may focus on expanding integrated emitter lifetimes, optimizing trap-and-elute modes for a wider range of sample types, and integrating advanced ion mobility interfaces. Scalability to even larger clinical cohorts and adoption of greener solvent systems may further enhance sustainability. Deep learning-driven data acquisition strategies could synergize with these robust hardware platforms to maximize proteome coverage.

Conclusion

The OptiSpray µPAC Neo HT and PepMap Neo 75 µm × 15 cm cartridges with FAIMS provide exceptional longevity and reproducibility for low-flow proteomics. Their integrated emitter design and automated positioning deliver consistent chromatographic and identification metrics across thousands of injections, making them well suited for large-scale studies in research and industrial laboratories.

References

Walker KL et al Extended lifespan of innovative column assemblies in low-flow ion sources Thermo Fisher Scientific 2025