At the intersection between chromatographic performance, ESI efficiency and instrument productivity: nano to capillary flow LC/MS on long μPAC Columns

Significance of the Topic

High-throughput proteomics is essential for large-scale protein analysis, reducing analysis time while maintaining depth of coverage. Optimizing the balance between chromatographic resolution, electrospray ionization efficiency, and instrument productivity enables faster workflows without sacrificing data quality.

Objectives and Study Overview

This study investigates the maximum throughput achievable using 110 cm Thermo Scientific µPAC Neo Plus columns operated at nano to capillary flow rates. It evaluates single-emitter configurations and a novel dual-column, single-emitter tandem setup to measure the impact of post-column dispersion and enhance sample processing rates up to 50 samples per day (SPD).

Methodology and Instrumental Setup

• Sample Preparation: HeLa cell digests prepared at 200 ng/µL in 0.1% TFA, 1% ACN, sonicated and vortexed.
• LC Configuration: Thermo Scientific Vanquish Neo UHPLC with either a single µPAC Neo Plus column and PepMap Neo trap in trap-and-elute mode or two µPAC Neo Plus columns in tandem via low-dispersion six-port valves.
• Flow Rates and Gradients: Tested flows from 200 to 1,000 nL/min for throughputs of 16, 30, 40 and 50 SPD; gradient lengths ranged from under 20 minutes to over 50 minutes.
• Mass Spectrometry: Orbitrap Exploris 240/480 in DIA mode with MS1 resolution 120k, MS2 resolution 30k, variable isolation widths (4–15 Th) and AGC settings.
• Data Analysis: Processed using Spectronaut 19 and Proteome Discoverer 3.3 with Chimerys, applying a 1% FDR filter.

Key Results and Discussion

• Optimal 1 µL/min flow for gradients under 30 minutes identified up to 6,814 protein groups at 50 SPD, with 90% of proteins quantified at CV < 20%.
• For longer gradients (> 50 minutes) at 200 nL/min, proteome coverage increased to a maximum of 8,556 protein groups at 16 SPD.
• Dual-column single-emitter tandem setup increased productivity by over 25%, reaching 6,848 identified protein groups at 50 SPD and a median CV of 7.2% across five columns.
• Column-to-column reproducibility was high, showing < 1% variation in identification rates and a median retention time CV of 0.99% for spiked peptides.

Advantages and Practical Applications

• Enhanced sample throughput without compromising sensitivity achieves rapid profiling suitable for large cohort studies.
• Trap-and-elute workflows minimize overhead, improving robustness and reducing cycle time.
• Tandem column configuration leverages low-dispersion valves to maintain chromatographic performance at high flow rates.
• Applicable for pharmaceutical QA/QC, biomarker discovery, and high-throughput clinical proteomics.

Future Trends and Potential Applications

• Integration with faster and more sensitive MS platforms to further boost throughput and coverage.
• Adoption of advanced data acquisition strategies such as parallel reaction monitoring for targeted workflows.
• Expansion into single-cell proteomics and large-scale clinical cohort analyses.
• Development of fully automated, multiplexed systems for routine high-throughput laboratory use.

Conclusion

The combination of long µPAC Neo Plus columns, optimized flow rates, and a dual-column single-emitter tandem configuration delivers outstanding throughput and depth in proteomic profiling. This approach supports rapid, reproducible analysis for diverse applications in research and industry.

References

• Zheng R, Rendl M, Valenta AC, Pynn C, Lin Y, Daniliuk M, Aydin E, van Ling R, Taujenis L, Decrop W, Samonig M, Morgenstern A. A dual-column, single-spray configuration for capillary and micro-flow LC-MS applications. Application note TN003314, Thermo Fisher Scientific; 2025.