Ultra-Fast Capillary-Flow LC-MS Profiling of Complex Biological Matrices: Applicable to Large Sample Cohorts

Importance of the topic

The growing demand for large‐scale protein profiling in biological fluids and cell lysates places strict requirements on analytical workflows to deliver both depth of coverage and high throughput. Traditional nanoLC‐MS methods offer excellent sensitivity but often lack robustness and speed when handling large sample cohorts. Capillary‐flow LC‐MS represents a promising compromise, combining the stability and reproducibility of microflow chromatography with the proteomic capabilities of modern high‐resolution mass spectrometers.

Objectives and study overview

This study aimed to develop, optimize, and validate a suite of ultra‐fast capillary‐flow LC‐MS methods capable of processing up to 180 samples per day. Using a Thermo Scientific™ UltiMate™ 3000 RSLCnano system coupled to a Q Exactive™ HF-X mass spectrometer, five distinct gradient lengths were evaluated (8, 14.4, 24, 48, 60 min) to balance throughput and proteome depth. Key performance metrics included MS utilization, chromatographic peak shape, and quantitative reproducibility across large cohorts.

Methodology and instrumentation

The workflow combined rapid sample loading, a trap‐and‐elute configuration, and precise gradient delivery. Key method features included:

• Five low‐flow gradient lengths: 8, 14.4, 24, 48, 60 min.
• Throughput ranging from 180 to 24 samples per 24 h; MS utilization of 75–95 %.
• Pre‐concentration on a trap cartridge for desalting and focusing at high flow.
• Fast autosampler injections (<1 min) with negligible carryover.
• Optimized gradient slopes and flow rates to preserve sensitivity at different run times.

Instrumentation Used

The analytical platform consisted of:

• Thermo Scientific™ UltiMate™ 3000 RSLCnano system in pre‐concentration mode.
• Trap cartridge RSLCnano pre‐concentration kit (P/N 6720.0310).
• 75 µm×150 mm EASY-Spray™ column (3 µm, P/N ES800).
• Thermo Scientific™ Q Exactive™ HF-X hybrid Quadrupole‐Orbitrap™ mass spectrometer with EASY-Spray™ source.

Main results and discussion

Protein identification performance scaled with gradient length. In 200 ng HeLa digest:

• 8 min run: >1 000 protein groups, >5 000 peptide groups.
• 60 min run: ~3 400 protein groups, ~24 000 peptide groups.

In crude plasma digests:

• 8 min run: ~150 protein groups, >1 000 peptide groups.
• 60 min run: >250 protein groups, ~2 700 peptide groups.

Reproducibility was demonstrated across >200 consecutive plasma injections in the 14.4 min method, showing retention time standard deviation <0.1 min and quantitative CV <20 % for 90 % of proteins. Peak shapes remained sharp (peak widths at half‐height 3–10 s) and symmetric (asymmetry ~1.15).

Benefits and practical applications

The developed capillary‐flow LC‐MS methods deliver:

• High throughput enabling large cohort studies.
• Robust performance with minimal maintenance and carryover.
• Sufficient proteome coverage for discovery and targeted assays.
• Broad dynamic range across five orders of magnitude.
• Compatibility with automated sample preparation and challenging matrices.

This workflow is well suited to clinical proteomics, biomarker research, QA/QC, and workflows requiring continuous, high‐volume sample analysis.

Future trends and applications

Further advances may include integration with robotic sample preparation, extension to deeper fractionation strategies, and implementation of data‐independent acquisition (DIA) workflows. Coupling capillary‐flow LC to emerging ultra‐high-field mass spectrometers could push both sensitivity and throughput further for routine large‐scale studies.

Conclusion

A novel suite of ultra‐fast capillary‐flow LC‐MS methods was established, offering a flexible balance between speed and depth. Validated on both HeLa and plasma digests, these methods support high‐throughput proteomic profiling with excellent robustness, reproducibility, and quantitative performance.

References

1. RSLCnano pre‐concentration nano LC kit. Ultimate 3000 RSLCnano Standard Applications Guide.
2. Boychenko A., Pynn C., van den Berg B., Arrey T.N., Baynham M., Decrop R., Ruehl M. High‐throughput capillary‐flow LC‐MS proteomics with maximum MS utilization. Thermo Fisher Scientific TN 72227.
3. Boychenko A., Meding S., Decrop W., Ruehl R., Swart R. Capillary‐flow LC‐MS: combining high sensitivity, robustness, and throughput. Thermo Fisher Scientific TN 72277.