Abundant Protein Depletion of Human Plasma Samples – A Reproducibility and Scaling Study

Significance of the Topic

The large dynamic range of protein concentrations in human plasma presents a key challenge for biomarker discovery. Highly abundant proteins mask low‐abundance species of diagnostic or prognostic relevance, limiting sensitivity and depth of proteomic analysis. Efficient, reproducible depletion of albumin, immunoglobulins and other high‐abundance targets enhances detection of clinically relevant biomarkers and improves quantitative accuracy.

Objectives and Study Overview

This study describes the development and evaluation of two novel immunoaffinity spin‐column resins, Top2 and Top12, designed to deplete the most abundant plasma proteins. Specific aims included:

• Optimization of ligand conjugation and resin formulation for Top2 (Albumin, IgGs) and Top12 (12 target proteins).
• Assessment of depletion efficiency, sample yield and reproducibility across varying sample loads.
• Comparison of depletion performance by label‐free LC‐MS, colorimetric peptide assays and ELISA.

Methodology and Instrumentation Used

Sample Preparation and Depletion

• Normal pooled human plasma (10 µL input per spin column) was incubated with Top2 or Top12 resins, followed by centrifugation to collect flow‐through.
• Triplicate biological replicates processed for each resin; protein concentration measured by BCA assay and Pierce peptide assay.
• Proteins precipitated, reduced/alkylated with TCEP/CAA, digested with trypsin, and desalted prior to LC‐MS.

Analytical Platforms

• LC‐MS performed on Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer coupled to Dionex Ultimate 3000 Nano LC with a 50 cm EASY‐Spray C18 column.
• Data processed using Proteome Discoverer 1.4 and Skyline 3.6.
• Depletion validated by ELISA for individual targets and by MS‐based quantitation of proteotypic peptides.

Main Results and Discussion

Depletion Efficiency and Specificity

• Both Top2 and Top12 achieved >95% removal of target proteins, as confirmed by ELISA and LC‐MS peak‐area analysis.
• Coefficient of variation (CV) for target depletion across triplicates was <1% for MS‐based measurements and <5% for peptide assays.

Proteome Coverage

• Relative to undepleted plasma, Top2 yielded a 10% increase and Top12 a 30% increase in unique protein group identifications.
• Non‐targeted protein recovery remained highly reproducible, with CVs <30% for biological replicates.

Correlation of Methods

• Good agreement observed between MS‐based depletion values and ELISA, demonstrating cross‐method validity.
• Optimization of post‐depletion handling (reduction, digestion, peptide recovery) proved critical for consistent quantitation.

Benefits and Practical Applications

Implementing Top2 and Top12 spin‐column depletion enables:

• Enhanced detection of low‐abundance biomarkers in plasma proteomics workflows.
• Streamlined sample preparation with minimal handling steps and disposable format.
• Scalable processing of small (10 µL) to moderate sample volumes with high reproducibility.
• Compatibility with label‐free quantitative LC‐MS platforms and orthogonal assays.

Future Trends and Applications

Anticipated developments include:

1. Expansion to multiplexed depletion targets and customizable resin panels for disease‐specific proteomics.
2. Integration with automated and high‐throughput workflows for clinical and industrial laboratories.
3. Adoption of advanced data‐independent acquisition (DIA) methods to further enhance depth and quantitation.
4. Use of AI‐driven data analysis to identify novel biomarkers from complex plasma datasets.

Conclusion

The newly developed Top2 and Top12 immunoaffinity spin columns deliver highly efficient and reproducible depletion of abundant plasma proteins. Their implementation increases proteome coverage, supports reliable quantitation of low‐abundance targets, and streamlines workflows for biomarker discovery and clinical proteomics.

References

• Snovida SI, Herting KE, Ganapathy R, et al. Abundant Protein Depletion of Human Plasma Samples – A Reproducibility and Scaling Study. Thermo Fisher Scientific; 2017.