Proteomic Analysis of Plasma – Sample Preparation and Multiplexing Workflows for Relative Quantitation

Significance of the Topic

The wide dynamic range of protein concentrations in human plasma poses a major barrier for biomarker discovery and accurate quantitation of low-abundance proteins. Effective removal of highly abundant proteins combined with multiplexed labeling enhances the detection of clinically relevant biomarkers in complex plasma matrices.

Objectives and Study Overview

This study aimed to develop and apply a streamlined workflow for comparative proteomic profiling of plasma samples from healthy donors and patients with pulmonary hypertension heart failure with preserved ejection fraction (PH-HFpEF). Key goals included:

• Achieving reproducible depletion of the top 14 abundant plasma proteins.
• Establishing a rapid, high-throughput sample preparation protocol for small plasma volumes.
• Implementing multiplexed Tandem Mass Tag (TMT) labeling for relative quantitation across multiple sample sets.
• Identifying differentially abundant proteins as potential PH-HFpEF biomarkers.

Methodology and Instrumentation

Plasma samples (10 µL each) were depleted of abundant proteins using Top14 affinity columns, followed by digestion with the EasyPep Mini MS Sample Prep Kit. Peptide-level labeling employed TMT10plex reagents plus an 11th channel, enabling five 11-plex sets. Each set combined individual patient and control samples with pooled and bridging channels. Post-labeling, high pH reversed-phase fractionation generated eight fractions per set.

Used Instrumentation:

• Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer with synchronous precursor selection (SPS) mode.
• 75 cm C18 column and three-hour gradient for LC separation.
• Thermo Scientific Proteome Discoverer 2.3 software for data processing and quantitation.

Main Results and Discussion

Across five 11-plex experiments, 1043 protein groups and 8580 peptide groups were identified with >90% digestion efficiency. Comparative analysis revealed significant differences between PH-HFpEF and control cohorts:

• Decreased abundance of serum albumin in PH-HFpEF patients, suggesting potential renal loss mechanisms.
• Elevated von Willebrand factor levels in the PH-HFpEF cohort, consistent with cardiovascular disease profiles.
• Marked reductions in IGF-binding protein complex acid-labile subunit and IGF-binding protein 3, indicating disrupted IGF signaling in disease.
• Complement C-4B and its binding protein showed no significant changes.

Volcano plots and bar charts confirmed reproducibility of depletion and quantitation across pooled and bridging channels, supporting workflow robustness.

Benefits and Practical Applications

The integrated depletion and EasyPep-based sample preparation workflow offers:

• High reproducibility in abundant protein removal (>95% target depletion).
• Efficient processing of low-volume plasma samples with minimal handling steps.
• Enhanced proteome coverage enabling detection of potential low-abundance biomarkers.
• Multiplexed TMT labeling for cost-effective relative quantitation across multiple cohorts.

This approach is well suited for clinical studies, biomarker verification, and translational research in cardiovascular and metabolic disorders.

Future Trends and Potential Applications

Advancements likely to further improve plasma proteomics include:

• Automation of depletion and digestion steps to increase throughput and reproducibility.
• Integration of deeper fractionation techniques or ion mobility separation for expanded proteome coverage.
• Application of data-independent acquisition (DIA) strategies to complement TMT-based quantitation.
• Implementation of longitudinal profiling for monitoring disease progression and treatment response.

Conclusion

The described workflow combining Top14 depletion, EasyPep sample preparation, TMT multiplexing, and Orbitrap Fusion analysis delivers reliable, high-throughput plasma proteomics data. Differential protein abundances observed between PH-HFpEF patients and controls highlight potential biomarker candidates and underscore the method’s utility for clinical proteomic investigations.

Reference

• Simon MA; Vanderpool RR; Nouraie M; Bachman TN; White PM; Sugahara M; Gorcsan J III; Parsley EL; Gladwin MT. Acute hemodynamic effects of inhaled sodium nitrite in pulmonary hypertension associated with heart failure with preserved ejection fraction. JCI Insight. 2016;1(18).
• Lai YC; Wang L; Gladwin MT. Insights into the pulmonary vascular complications of heart failure with preserved ejection fraction. J Physiol. 2018;597(4).