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

Importance of the Topic

High-throughput bottom-up proteomics demands rapid and reproducible peptide separation to maximize proteome coverage in large sample cohorts. Advances in LC column design directly influence analysis speed, peak capacity, and robustness. The newly engineered micro pillar array column (µPAC Neo High Throughput) addresses these needs by combining high efficiency with low back pressure, enabling both nano and capillary flow regimes on a compact 5.5 cm format.

Objectives and Study Overview

This study aimed to evaluate the performance of the Thermo Scientific™ µPAC™ Neo 5.5 cm High Throughput column featuring rectangular pillar arrays, comparing it to traditional packed emitter columns under various flow rates, gradient lengths, and sample loads. Key goals included assessing peptide and protein identification numbers, peak widths, and retention time reproducibility for workflows targeting 100–180 samples per day (SPD).

Applied Methodology and Instrumentation

The analytical platform consisted of a Vanquish™ Neo UHPLC system coupled to an Orbitrap Exploris™ 480 mass spectrometer operating in data-dependent acquisition mode. Key steps included:

• Sample Preparation: Thermo Scientific™ Pierce™ HeLa Protein Digest Standard diluted to 200 ng/µL in 0.1 % formic acid with PRTC calibration peptides at 100 fmol/µL.
• Column Setup: µPAC Neo High Throughput column (5.5 cm) heated to 50 °C in a Sonation oven; outlet connected to EASY-Spray™ emitters (15 µm or 20 µm ID).
• Flow Regimes: Explored nano flow (300 nL/min) and capillary flows (0.2–2.5 µL/min) with gradient times from 5 to 30 min.
• Data Analysis: Processed with Proteome Discoverer 3.0 and CHIMERYS™; peptide peak widths quantified via apQuant; FDR set at 1 % for peptides and proteins.

Main Results and Discussion

Radially elongated rectangular pillars enhanced separation efficiency, achieving full width at half maximum (FWHM) below 1.5 s even at short (5 min) gradients. Key findings include:

• Optimal Peptide IDs: Under 5 min gradients, capillary flows of 1–1.5 µL/min yielded up to 30 % more peptide identifications compared to packed emitter columns, across sample loads of 50 ng–1 µg.
• Gradient Dependence: Longer gradients (30 min) increased overall protein and peptide identifications, with nano flow favoring narrower peaks at extended times, while capillary flow excelled for short gradients.
• Reproducibility: Retention time RSD for PRTC peptides remained below 1 % across three µPAC columns and multiple runs, outperforming packed emitters (>1.4 % RSD for early eluting peptides).

Benefits and Practical Applications

The µPAC Neo High Throughput column delivers:

• High sample throughput (100–180 SPD) without sacrificing resolution.
• Enhanced proteome coverage, identifying up to 7 % more proteins and nearly 30 % more peptides.
• Robust column-to-column and run-to-run consistency essential for large-scale studies.
• Flexibility to shift between nano and capillary flows for diverse proteomic workflows.

Future Trends and Potential Applications

Building on this technology, future directions include:

• Integration with ultrafast gradient systems to push beyond 180 SPD.
• Customized pillar geometries for targeted applications such as post-translational modification analysis.
• Coupling with emerging high-field MS platforms for deeper proteome coverage.
• Automation in clinical and industrial QC settings, leveraging reproducibility and throughput.

Conclusion

The Thermo Scientific™ µPAC™ Neo High Throughput column demonstrates a significant advancement in high-throughput LC-MS proteomics. Its unique pillar design and short channel length enable rapid separations, improved peptide coverage, and exceptional reproducibility, making it a valuable tool for large-scale proteomic studies.

Reference

Data derived from Thermo Fisher Scientific application note on µPAC Neo High Throughput column performance.