Novel tandem nano and capillary flow LC-MS-based approach for facile 24/7 proteome profiling with near 100% MS utilization

Importance of the Topic

Proteome profiling at nano and capillary flow rates is critical for sensitive and comprehensive analysis of complex biological samples. However, conventional single‐column LC-MS systems suffer from low mass spectrometer utilization due to sample injection, column equilibration, and wash overheads. Maximizing instrument duty cycles enhances throughput and data quality in both research and routine applications.

Objectives and Study Overview

This work presents a tandem direct injection tandem LC-MS workflow designed to alternate two analytical columns, aiming to achieve near-100% MS utilization for continuous 24/7 proteome profiling. Performance is evaluated at nano-flow (0.3 µL/min, 65-min gradient) and capillary-flow (1.5 µL/min, 8-min gradient) conditions to balance depth and speed.

Methodology and Instrumentation

• Dual‐column separation on a Thermo Scientific Vanquish Neo UHPLC system with look-ahead injections and automated column switching.
• Double Barrel Column Oven (Sonation) enabling simultaneous interfacing of two fused‐silica columns without post-column splitting.
• Orbitrap Exploris 480 high-resolution accurate‐mass mass spectrometer operating in DIA and DDA modes.
• NanoViper fluidic connections and nanoSpray Flex ion source ensuring stable, low‐dead‐volume electrospray ionization.

Main Results and Discussion

• Achieved 94% MS utilization with the 65-min nano-flow method and 79% utilization with the 8-min capillary-flow method during uninterrupted operation.
• Sample throughput reached 22 injections per day in nano mode and up to 180 injections per day in capillary mode.
• Retention time variability remained below 0.5 min across columns, and peak area reproducibility supported accurate quantification.
• The workflow adapts easily to various sample types, including phosphoproteome and TMT-labeled peptide analyses, by fine-tuning gradients and wash cycles.

Benefits and Practical Applications

• Maximizes utilization of MS acquisition time, reducing idle periods.
• Maintains high chromatographic resolution and consistent quantification.
• Suitable for both in‐depth discovery proteomics and high‐throughput screening in pharmaceutical and clinical laboratories.

Future Trends and Potential Applications

Advances in automation, method customization, and real-time feedback control may further enhance tandem injection workflows. Integration with targeted quantification protocols and microfluidic column designs could expand applications in biomarker validation and clinical proteomics.

Conclusion

The tandem direct injection LC-MS workflow on the Vanquish Neo platform, coupled with a double‐barrel ESI source and HRAM detection, delivers near‐100% MS utilization and excellent reproducibility. This approach outperforms traditional single‐column methods and provides a robust solution for continuous, high‐throughput proteome profiling.

References

1. Zheng R., Pynn C., et al. New Double Barrel ESI Source and Novel Tandem NanoLC-MS for 24/7 Proteome Profiling with near 100% MS Utilization. Thermo Scientific TN73671.
2. Boychenko A., Pynn C., et al. High-throughput tandem capillary-flow LC-MS for maximum MS utilization. Thermo Scientific TN72827.