High-throughput analysis with improved proteome coverage using new designed micro pillar array column (μPAC)

Significance of the Topic

Proteomic LC-MS based methodologies are pivotal for analyzing complex biological samples. High-resolution separation reduces sample complexity prior to mass spectrometry analysis, driving improvements in sensitivity and throughput.

Objectives and Study Overview

This study introduces a novel 5.5 cm µPAC Neo High Throughput column featuring rectangular micro pillar arrays with radially elongated pillars. The goal is to assess its performance in bottom-up proteomics workflows under short gradient and high flow rate conditions compared to traditional packed emitter columns.

Methodology and Instrumentation

All experiments were performed on a Thermo Scientific Vanquish Neo UHPLC system operated in direct injection mode coupled to an Orbitrap Exploris 480 mass spectrometer in DDA mode. The µPAC Neo column was heated to 50 °C and interfaced with EASY-Spray Nano & Capillary Emitters (15 µm or 20 µm I.D.). A HeLa protein digest standard (200 ng) spiked with PRTC peptides (100 fmol/µL) was analyzed using gradient lengths of 5–30 min at flow rates ranging from 0.2 to 2.5 µL/min. Data processing was conducted using Proteome Discoverer v3.0 with CHIMERYS, applying a 1% FDR and apQuant for peak width determination.

Main Results and Discussion

The µPAC Neo High Throughput column achieved full width at half maximum (FWHM) values below 1.5 s even with 5 min gradients, enabling capillary flow rates (1–2.5 µL/min) without sacrificing resolution. Optimal peptide and protein identifications were observed at 1–1.5 µL/min in short gradients, while longer 30 min methods benefited from nano flow (300 nL/min) for deeper coverage. Compared to packed emitter columns (150 µm×50 mm, 1.7 µm) under 180 and 100 samples per day methods, the µPAC Neo column identified 2.6–7.2% more proteins and 8.1–28.6% more peptides, with improved retention time reproducibility (RSD <1%) across columns and runs.

Benefits and Practical Applications

• Enables high-throughput workflows (up to 180 SPD) with short gradients and capillary flows
• Delivers enhanced proteome coverage and narrower chromatographic peaks
• Reduces instrument backpressure and supports flexible flow programming
• Offers robust run-to-run and column-to-column reproducibility

Future Trends and Opportunities

Further optimization of micro pillar geometries may push throughput and depth even higher. Integration with real-time data acquisition strategies and AI-driven gradient design could enhance performance. Potential applications include clinical proteomics, pharmaceutical quality control, and high-content biomarker discovery in reduced sample amounts, including single-cell analyses.

Conclusion

The new µPAC Neo High Throughput column demonstrates significant gains in speed, sensitivity, and reproducibility for LC-MS proteomics. Its ability to maintain high resolution at capillary flow rates and short gradients makes it a versatile platform for both routine and deep proteome profiling.

References

Sun X, Lin Y, Op de Beeck J, Robson BH, Silveira JA, Jacobs P, Swart R, Lin S. High-throughput analysis with improved proteome coverage using new designed micro pillar array column (µPAC). Thermo Fisher Scientific Technical White Paper; 2023.