High throughput, routine and comprehensive proteome analysis using a μPAC HPLC column based capillary-flow LC-MS workflow

Importance of the Topic

A robust, high‐throughput and highly reproducible proteome analysis workflow is essential for large‐scale quantitative studies in research, industrial and clinical laboratories. Optimizing chromatographic separation and mass spectrometric sensitivity while balancing sample throughput remains a key challenge.

Objectives and Study Overview

This work evaluates micro pillar array chromatography columns produced by microfabrication (μPAC) for three proteomics scenarios: high throughput (12 samples per day comprehensive vs up to 65 samples per day), routine operation and in‐depth analysis. HeLa cell tryptic digests (2 μg injections) are separated across gradients of 15 to 120 minutes at capillary flow rates from 2 to 10 μL per minute.

Methodology and Instrumentation

Micro pillar array columns are etched from silicon wafers to produce highly uniform, superficially porous pillars (5 μm diameter, 28 μm length, 50 cm bed length). Columns operate at 1 to 15 μL per minute under backpressures up to 350 bar. Separation is performed in reversed‐phase mode with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in 80% acetonitrile). Three non‐linear gradient methods (17.5, 54 and 108 min) are applied on a capillary LC‐MS/MS system with direct injection and optimized sample pickup.

Main Results and Discussion

• High throughput mode (10 μL/min, 17.5 min gradient): 65 samples per day with 83% MS utilization; average peptide peak width (FWHM) 3.97 s; retention time CV 0.12% (0.75 s); identification of ~1 685 peptide groups and 512 protein groups.
• Routine mode (5 μL/min, 54 min gradient): 24 samples per day with 89% MS utilization; average FWHM 8.25 s; retention time CV 0.08% (1.13 s); ~5 741 peptide groups and 1 387 protein groups per day.
• Comprehensive mode (2 μL/min, 108 min gradient): 12 samples per day with 84% MS utilization; average FWHM 11.58 s; retention time CV 0.07% (1.81 s); ~9 448 peptide groups and 2 175 protein groups per day; peak capacity up to 335 at peak base.

Practical Benefits and Applications

The μPAC column platform delivers exceptional column‐to‐column reproducibility and flexible flow rate operation, allowing end users to tailor LC‐MS methods for diverse workflows: rapid screening, routine profiling and in‐depth proteome mapping. High MS time utilization and narrow peak widths increase data productivity and quantitation precision.

Future Trends and Opportunities

• Integration with data‐independent acquisition and advanced bioinformatics for deeper proteome coverage.
• Automation of gradient optimization using machine learning to further enhance throughput and reproducibility.
• Expansion of microfabrication techniques to novel stationary phase geometries and materials for improved separation power.
• Application in clinical and biopharma QA/QC for standardized, high‐throughput biomarker validation.

Conclusion

Micro pillar array columns offer a highly versatile platform for capillary‐flow LC‐MS proteomics. By selecting appropriate flow rates and gradient lengths, researchers can achieve outstanding reproducibility, sensitivity and throughput across diverse proteomic applications.

Used Instrumentation

• Thermo Scientific μPAC HPLC column, 50 cm × 28 μm pillar length
• Capillary LC‐MS/MS system with direct injection mode
• Gradient pump delivering 1–15 μL/min, max pressure 350 bar

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