Novel ion source with integrated separation column and replaceable emitters facilitates high-throughput multiplexed analysis

Importance of the Topic

The reliability and throughput of targeted proteomics workflows depend heavily on stable ionization and reproducible chromatography at low flow rates. Advances in ion source design that simplify setup, reduce variability between users, and maintain spray stability are crucial for high-throughput multiplexed analyses in both research and clinical laboratories.

Objectives and Study Overview

This study evaluated a new OptiSpray ion source, which integrates a replaceable separation column cartridge with an automated emitter positioning routine, on the Thermo Scientific Stellar MS platform. The goal was to determine whether this configuration enhances sensitivity, reproducibility, and ease of use during Parallel Reaction Monitoring (PRM) of a 57-protein Health Surveillance Panel (HSP). Throughputs of 100, 144, and 180 samples per day (SPD) were compared against a conventional EASY-Spray source.

Methodology and Instrumentation

PRM assays were developed using 83 stable isotope-labeled (SIL) peptides spiked into 50 ng of pooled plasma digests. Three throughput methods (100, 144, 180 SPD) were evaluated. An automated “capillary flow quick optimization” routine determined the optimal emitter position based on ion signal. Key ion source parameters included:

• Positive ion voltage: 1,800 V
• Sheath gas flow: 10 arb units
• Sweep gas flow: 0 arb units
• Ion transfer tube temperature: 250 °C
• Cartridge temperature: 45 °C

Used Instrumentation

• Thermo Scientific Stellar mass spectrometer (dual-pressure linear ion trap/triple quadrupole hybrid)
• Thermo Scientific Vanquish Neo UHPLC system
• OptiSpray ion source with integrated PepMap Neo 150 µm × 15 cm column cartridge
• Skyline and PRM Conductor software for data analysis

Main Results and Discussion

The OptiSpray source delivered higher ionization efficiency and lower coefficients of variation compared to the EASY-Spray source across all throughputs. Key findings:

• Automated emitter positioning reduced user-to-user variability and simplified setup.
• Across 10 technical replicates, OptiSpray showed consistent signal intensities, increased data points per peak, and stable retention times.
• Limits of detection (LOD) reached below 0.05 fmol for many peptides, with linear quantification (R² > 0.9) from 0.002 to 120 fmol.
• Switching cartridges preserved the stored optimal emitter position, supporting robust reproducibility in long sequences.

Benefits and Practical Applications

The integrated cartridge/emitter design and hands-off optimization deliver:

• Rapid, plug-and-play installation of low-flow columns without manual alignment.
• Reduced downtime and maintenance through controlled emitter positioning.
• Enhanced quantitative performance for biomarker validation, clinical proteomics, and high-throughput screening.

Future Trends and Possibilities

Further developments may include even higher-density multiplexing, real-time adaptive optimization, and broader compatibility with alternative column chemistries. Integration with machine-learning-driven tuning routines could further streamline method setup and system diagnostics. Expansion to other omics workflows (metabolomics, lipidomics) is also anticipated.

Conclusion

The OptiSpray ion source on the Stellar MS platform offers a robust, user-friendly solution for high-throughput PRM analyses. Its integrated cartridge design, automated emitter alignment, and stable spray performance enable sensitive, reproducible quantification of multiplexed peptide panels with minimal manual intervention.

References

• Fu Q., Walker K.L., Zheng R., Remes P.M., Heil L., Jacob C., Boeser C.L., Van Eyk J.E., Hassell K.M. Novel Ion Source with Integrated Separation Column and Replaceable Emitters Facilitates High-Throughput Multiplexed Analysis. Thermo Fisher Scientific; 2025.
• bioRxiv. 2025. DOI: 10.1101/40161722. PMID: 40161722.