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Updated: May 6, 2026
Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Assessment of reproducibility in depletion and enrichment workflows for plasma proteomics using label-free
Amirmansoor Hakimi1, Janica Auluck, George D D Jones
1Department of Cancer Studies and Molecular Medicine, RKCSB, University of Leicester, Leicester, UK.
Insights
Both immunodepletion and enrichment methods reproducibly quantify plasma proteins for proteomics. Enrichment identifies more proteins but requires larger sample volumes, while immunodepletion is suitable for limited samples.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biotechnology
Background:
- Plasma proteomics requires removing abundant proteins to detect low-abundance ones.
- Immunodepletion and enrichment are common methods for abundant protein removal.
- Label-free analysis enables quantitative assessment without chemical labels.
Purpose of the Study:
- To quantitatively assess the reproducibility of immunodepletion and enrichment methods for plasma proteomics.
- To compare the effectiveness of these two predominant protein removal techniques.
- To evaluate their suitability for biomarker studies.
Main Methods:
- Human plasma samples were analyzed using nano-LC-data-independent acquisition MS.
- Label-free quantitative analysis was employed.
- Reproducibility was assessed using 18 candidate proteins and inter-day variability.
Main Results:
- Both immunodepletion and enrichment showed reproducible results with residual standard deviation below 20%.
- Enrichment identified more proteins (272) compared to immunodepletion (200).
- Both methods met analytical standards for protein quantitation reproducibility.
Conclusions:
- Both immunodepletion and enrichment are reproducible and suitable for plasma proteomics and biomarker studies.
- Enrichment is advantageous for identifying a greater number of proteins when sample volume is not limited.
- Immunodepletion is a viable option when dealing with limited sample volumes.
Abstract:
Quantitation in plasma-based proteomics necessitates the reproducible removal of highly abundant proteins to enable the less abundant proteins to be visible to the mass spectrometer. We have evaluated immunodepletion (proteoprep20) and enrichment (Bio-Rad beads), as the current predominant approaches. Label-free analysis offers an opportunity to estimate the effectiveness of this approach without incorporating chemical labels. Human plasma samples were used to quantitatively assess the reproducibility of these two methods using nano-LC-data-independent acquisition MS. We have selected 18 candidate proteins and a comparison of both methodologies showed that both of the methods were reproducible and fell below 20% residual SD. With the same candidate proteins, individual inter-day variability for the samples was also processed, allowing us to monitor instrument reproducibility. Overall, a total of 131 proteins were identified by both methods with 272 proteins identified by enrichment and 200 identified by immunodepletion. Reproducibility of measurements of the amount of protein in the processed sample for individual proteins is within analytically acceptable standards for both methodologies. This enables both methods to be used for biomarker studies. However, when sample is limited, enrichment is not suitable as larger volumes (>1.0 mL) are required. In experiments where sample is not limited then a greater number of proteins can be reliably identified using enrichment.
