Routine proteome analysis using 50 cm μPAC columns

Significance of the Topic

Bottom up proteomics relies on high performance nano liquid chromatography mass spectrometry to separate complex peptide mixtures prior to MS analysis. The development of robust, high capacity columns enhances proteome coverage while enabling higher throughput. Optimizing separation reduces run times and improves identification depth in routine and deep proteome studies.

Study Objectives and Overview

This study evaluates a 50 cm micro pillar array column µPAC for routine proteome analysis and benchmarks its performance against two 15 cm packed bed columns. Using triplicate gradients at 30, 60 and 90 minutes and flow rates of 300 and 1000 nL per minute, the goal was to assess separation performance, throughput and proteome coverage.

Methodology and Instrumentation

Sample Preparation

• Thermo Scientific Pierce HeLa Protein Digest Standard reconstituted to 500 ng per µL
• Spiking with Pierce Retention Time Calibration peptides at 50 fmol per µL

Instrumentation

• Thermo Scientific nano liquid chromatography system at 50 °C
• 50 cm µPAC column and two 15 cm packed bed columns
• Flow rates: 300 and 1000 nL per minute
• Gradient: non linear from 1 to 50 percent acetonitrile in 30 60 or 90 minutes
• High resolution mass spectrometer with New Objective PicoTip emitter
• Data processed with Proteome Discoverer software against UniProt human database at 0.1 percent FDR

Main Results and Discussion

Flow Resistance and Void Time

• µPAC column backpressure at 300 nL per minute was 40 bar versus 249 and 296 bar for packed bed columns
• Void time extended to 18.1 minutes at 300 nL per minute and shortened to 5.4 minutes at 1000 nL per minute

Peak Shape and Capacity

• Average peak widths at four sigma ranged from 0.13 to 0.22 minutes on µPAC compared to broader peaks on packed beds
• Peak capacity exceeded 200 for 30 minute gradients at high flow rates, outperforming conventional columns across all gradient lengths

Proteome Coverage

• With 60 minute gradients the µPAC column identified over 1700 protein groups and 6100 peptide groups
• This represents approx 40 percent increase in protein identifications and 60 percent in peptide identifications compared to packed bed columns

Benefits and Practical Applications

• Low backpressure enables use of longer columns or higher flow rates without exceeding instrument limits
• Enhanced peak capacity and sharper peaks translate into deeper proteome coverage in shorter runs
• Improved reproducibility from microfabricated pillar arrays supports high throughput routine analyses in research and QA QC environments

Future Trends and Applications

Advances in microfabrication may allow even longer or more complex pillar geometries to further boost peak capacity. Integration with faster mass spectrometers could leverage high flow capabilities for sub thirty minute proteome profiling. Automation of µPAC column production and standardization will support broader adoption in industrial and clinical laboratories.

Conclusion

The 50 cm µPAC column demonstrates superior chromatographic performance over conventional packed bed columns in routine proteome analysis. Its low backpressure, high peak capacity and improved proteome coverage at both low and high flow rates make it a valuable tool for increasing throughput without compromising depth of analysis.

References

• W De Malsche et al Experimental Study of Porous Silicon Shell Pillars in Liquid Chromatography Anal Chem 2008 80 5391–5400
• W De Malsche et al Realization of One Million Theoretical Plates Using Very Long Pillar Array Columns Anal Chem
• U D Neue Peak capacity in unidimensional chromatography J Chromatogr A 2008 1184 107–130