A highly sensitive and robust 150 μm column to enable high-throughput proteomics

Significance of the topic

Advances in proteomics demand chromatography solutions that combine high sensitivity, throughput, and robustness to handle complex biological samples and large cohorts. The development of a stable capillary LC column supports reliable peptide separation, consistent retention, and reproducible quantitation, which are essential for translational research and QA/QC in industrial and clinical settings.

Study objectives and overview

This study evaluates the Thermo Scientific EASY-Spray PepMap C18 capillary column (2 μm particles, 15 cm × 150 μm ID) for its reproducibility, robustness, and proteome coverage when coupled to a Q Exactive HF-X Orbitrap mass spectrometer using a data-dependent acquisition plus workflow. The aim is to demonstrate consistent performance over hundreds to a thousand injections across low-flow and high-flow regimes.

Methodology and instrumentation

• Sample preparation Standards: HeLa protein digest and peptide retention time calibration mixture (PRTC).
• Chromatography System: Thermo Scientific UltiMate 3000 RSLCnano in preconcentration mode with a 300 μm × 5 mm trap in back-flush, connected to a 15 cm × 150 μm EASY-Spray column at 40 °C.
• Mobile phases: A = water with 0.1% formic acid, B = acetonitrile with 0.1% formic acid. Low-flow method at 1.2 μL/min, 60 min gradient; high-flow method at 3 μL/min, 15 min gradient.
• Mass spectrometry: Q Exactive HF-X Orbitrap in DDA+ mode. Full MS at 120,000 resolution, 3e6 AGC target, 10 ppm mass tolerance; MS/MS at 7,500 resolution, 1e5 AGC target, 0.02 Da fragment tolerance; static carbamidomethylation and dynamic methionine oxidation. Data processed with Proteome Discoverer 2.2 using label-free quantitation nodes.

Main results and discussion

• Column-to-column reproducibility showed inter-column retention time RSD <3% and back pressure variation <3% across three columns.
• Intra-column stability demonstrated run-to-run retention RSD <0.5% over 20 injections.
• Proteome coverage of over 4,500 proteins and ~30,000 peptides with identification RSD <2% across columns.
• Column robustness assessed by monitoring three PRTC peptides over 400 injections, yielding retention RSDs between 1.56% and 2.73%.
• Long-term performance over 1,000 injections maintained proteome coverage and quantitation precision, with protein group RSD <5% including proteins at RSD <20%.

Benefits and practical applications

• Plug-and-spray column format reduces setup time and risk of leaks.
• Balanced sensitivity and robustness enable both deep proteome profiling and rapid targeted assays.
• High sample throughput and long column lifetime support large-cohort studies with minimal maintenance.
• Enhances label-free quantitation precision for monitoring subtle biological changes.

Future trends and possibilities

Further integration of microfluidics and novel packing materials will push sensitivity and durability. Enhanced acquisition strategies such as real-time search DDA and data-independent workflows may increase proteome depth. Machine learning-driven data analysis and predictive maintenance could further improve reproducibility and uptime in high-throughput proteomics operations.

Conclusion

The 15 cm × 150 μm EASY-Spray PepMap C18 column, paired with the Q Exactive HF-X and DDA+ workflow, delivers highly reproducible, robust, and sensitive proteomic separations. Its consistent performance over extensive injection series makes it well suited for standardized, large-scale proteomics and translational research applications.

References

• Thermo Fisher Scientific 2017 Pushing the leading edge in protein quantitation Integrated precise and reproducible proteomic workflows
• Thermo Fisher Scientific 2017 Increased sample throughput with a capillary EASY-Spray column