A tandem capillary and micro-flow LC workflow for high-throughput quantitative proteomics at near 100% mass spectrometer utilization

Significance of the topic

A robust high-throughput LC-MS platform that sustains near complete mass spectrometer utilization is essential for large-scale proteomic applications in research, clinical analysis, and quality control. By combining capillary and micro-flow chromatography with tandem direct injection, sample throughput is greatly increased without compromising sensitivity or reproducibility.

Objectives and Study Overview

This study evaluates a novel tandem capillary and micro-flow LC workflow on the Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 240 mass spectrometer. Key goals include:

• Maximize MS utilization across various flow rates and column dimensions
• Assess proteome depth and quantitation precision at high sample throughput
• Characterize retention time and abundance reproducibility
• Evaluate column carryover under extended washing cycles

Methodology and Instrumentation

The workflow integrates two parallel columns sharing a single electrospray emitter in a tandem direct injection (TDI) format. Method steps are executed by the system driver to align loading, washing and equilibration on the inactive column while acquisition proceeds on the active column. Capillary-flow methods (150 µm ID, flow rates 0.25–1.5 µL/min) and micro-flow methods (300 µm–1 mm ID, 1–15 µL/min) were tested at throughputs ranging from 48 to 277 samples per day.

Main Results and Discussion

• MS utilization exceeded 90 % across all tested configurations, reaching 98 % at moderate throughputs.
• At 180 SPD using a 150 µm × 150 mm column, 3 111–4 281 protein groups were identified from 50–1 000 ng HeLa digest in data independent acquisition mode, with median retention time RSD of 4 % and protein abundance RSD of 6 %.
• Micro-flow at 1.5–3.8 µL/min yielded 3 111–4 281 protein groups at 180 SPD with CV < 20 % for over 3 500 proteins.
• Increasing column bore to 300 µm × 15 cm enabled 240 SPD with 2 282 proteins identified from 200 ng and 91 % MS utilization.
• A 5 cm column at 1 mm ID achieved 277 SPD and 2 400 protein groups from 1 µg digest.
• Extended wash cycles reduced column carryover to ~0.03 % for 200 ng injections, without adding overhead time.

Benefits and Practical Applications

The TDI workflow delivers:

• High sample throughput with minimal idle time for the mass spectrometer
• Improved robustness and emitter lifetime due to consolidated spraying
• Consistent quantitative performance across columns and runs
• Low carryover suitable for demanding clinical or industrial assays

Future Trends and Perspectives

Emerging developments may focus on integrating higher-capacity columns, further automation of method setup, and real-time feedback control to sustain peak performance. The combination of capillary and micro-flow tiers could be extended to single-cell proteomics and large-cohort studies, while advances in mass spectrometer speed and data processing software will continue raising quantitative depth and throughput.

Conclusion

This study demonstrates that a tandem capillary/micro-flow LC workflow on the Vanquish Neo system can achieve near 100 % MS utilization, high proteome coverage, excellent reproducibility, and minimal carryover at up to 277 samples per day. The platform is well suited for high-throughput proteomics in research and regulated environments.

References

1. Zheng R et al Thermo Fisher Scientific Technical Note 003335 2024
2. Bian Y et al Robust reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS Nature Communications 2020 11 157
3. Zheng R et al Thermo Fisher Scientific Technical Note 000138 2021