Reducing Cycle Time for Affinity Removal of High-Abundant Proteins in Human Plasma

Importance of the topic

The human plasma proteome spans protein concentrations that vary over ten orders of magnitude. High-abundant proteins can obscure the detection of low-abundance biomarkers by mass spectrometry, making their efficient removal essential for sensitive proteomic analyses.

Objectives and study overview

This study aims to accelerate the depletion of the top 14 abundant proteins from human plasma by integrating alternating column regeneration on Agilent LC systems. By running one affinity column while regenerating the other, the workflow seeks to reduce cycle time and maintain high reproducibility.

Methodology and used instrumentation

The depletion was performed using Agilent’s Multiple Affinity Removal System (MARS) columns (4.6×50 mm) and bio-inert flow paths on the 1260 Infinity Quaternary LC paired with the 1290 Infinity Flexible Cube. A Quick-Change Bio-inert 2-position/10-port valve facilitated solvent routing. Plasma samples were processed with PBS dilution, affinity capture, and elution steps, employing a low flow rate (0.125 mL/min) for antigen binding and a higher rate (1 mL/min) for elution. Alternating regeneration cycles leveraged the 1290 piston pump to re-equilibrate columns concurrently with depletion runs.

Main results and discussion

Implementing alternating regeneration achieved a 32 % reduction in total cycle time. Over multiple runs, retention time precision remained below 0.13 % RSD and peak area precision under 1.04 % RSD across both high- and low-abundance fractions. These metrics demonstrate excellent intra- and inter-column reproducibility.

Benefits and practical applications

• Increases sample throughput by minimizing idle time between runs.
• Ensures reproducible depletion performance via a fully bio-inert flow path.
• Adapts to routine proteomic workflows requiring consistent removal of abundant plasma proteins.

Future trends and applications

Advancements may include automated multiplexed columns for even higher throughput, integration with online mass spectrometry for real-time analysis, and expansion to broader panels of affinity targets to enhance biomarker discovery in clinical proteomics.

Conclusion

Alternating column regeneration on Agilent’s bio-inert LC platforms substantially reduces cycle time while preserving high reproducibility in high-abundant protein depletion, offering a robust solution for advanced plasma proteomics.

References

1. Anderson L. Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J Physiol. 2005;563(Pt 1):36–60.
2. Agilent Technologies. Human Serum and Plasma Protein Depletion – Novel High-Capacity Affinity Column for the Removal of the “Top 14” Abundant Proteins. Application Note 5989-7839EN; 2008.
3. Agilent Technologies. Affinity removal of the 14 most abundant proteins in human plasma using the Agilent 1260 Infinity Bio-inert LC System. Application Note 5991-3207EN; 2013.