Multi-site assessment of precision and reproducibility of high-throughput capillary-flow LC-MS proteome profiling with novel ultra-high-pressure LC coupled to HRAM MS

Importance of the Topic

The adoption of high-throughput proteome profiling addresses the growing demand for rapid, sensitive, and reproducible analysis of large sample sets in research and quality control. By optimizing LC-MS workflows to reduce overhead and maximize instrument utilization, laboratories can achieve higher throughput without sacrificing data quality.

Objectives and Study Overview

This multi-site study evaluated the precision and reproducibility of five capillary-flow LC-MS methods using the Thermo Scientific Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 mass spectrometer. The methods targeted bottom-up proteome profiling of a HeLa digest standard, aiming to process up to 180 samples per day with a trap-and-elute workflow and to assess consistency across three independent laboratories.

Materials and Methods

The workflows comprised five total cycle times: 8, 14.4, 24, 48, and 60 minutes. All analyses used a 75 μm I.D. x 150 mm, 2 μm EASY-Spray PepMap Neo column with a constant flow rate of 300 nL/min. HeLa digest samples spiked with PRTC peptides were analyzed. Data processing employed Proteome Discoverer 2.5 with Sequest HT and INFERYS rescoring for peptide and protein identification.

Instrumentation Used

• Vanquish Neo UHPLC system (nano-, capillary-, and micro-flow)
• Orbitrap Exploris 480 high-resolution accurate-mass mass spectrometer
• EASY-Spray PepMap Neo column (75 μm I.D. x 150 mm, 2 μm particles)

Main Results and Discussion

The optimized methods produced median peak widths (FWHM) of 2 to 11 seconds for PRTC peptides, ensuring sharp chromatographic separation even in sub-15 minute gradients. MS utilization reached up to 95% by overlapping gradient runs with sample loading and column equilibration.

• Column-to-column reproducibility: ≤2% RSD for peptide and protein counts within each method; ≤6% RSD across three column sets on the same hardware.
• Retention time precision: stable retention times with <0.1% variation over 100 injections on the 14.4-minute method.
• Site-to-site reproducibility: ≥72% overlap in protein identifications across three laboratories for all cycle times.

Benefits and Practical Applications

This platform enables high-throughput, robust proteome analysis with minimal downtime, making it ideal for large-cohort studies in biomarker discovery, biopharmaceutical QA/QC, and clinical research. The trap-and-elute configuration simplifies workflow and enhances data consistency.

Future Trends and Opportunities

• Automated sample preparation integration to further boost throughput.
• Implementation of multiplexed quantitation strategies for deeper proteome coverage.
• Extension to diverse biological matrices, including clinical fluids and single-cell proteomics.

Conclusion

The Vanquish Neo UHPLC system paired with the Orbitrap Exploris 480 delivers rapid, sensitive, and reproducible bottom-up proteome profiling across multiple laboratories. This high-throughput approach effectively overcomes traditional LC-MS bottlenecks and supports scalable proteomic investigations.

References

No external literature cited.