An orthogonal extraction workflow for high-depth plasma proteomics with metabolomic compatibility from low...

Area of Science:

• Biochemistry
• Proteomics
• Metabolomics

Background:

• Plasma proteomics is crucial for biomarker development but faces challenges like low-abundance protein detection and limited molecular context.
• Current methods struggle with deep proteome coverage and integrating metabolomics due to separate sample preparation steps.
• These limitations hinder the clinical translation of protein biomarkers.

Purpose of the Study:

• To develop an orthogonal extraction workflow for enhanced plasma proteomic profiling and integrated metabolomics from small sample volumes.
• To overcome technical barriers limiting deep proteome coverage and metabolite stability in combined omics studies.
• To improve the efficiency and accuracy of biomarker discovery by integrating proteomic and metabolomic data.

Main Methods:

• Developed an orthogonal extraction workflow using a ternary solvent mixture (TSM) and perchloric acid (PCA) precipitation.
• Combined TSM and PCA for reproducible yet non-overlapping protein subset recovery.
• Integrated metabolomics analysis with enhanced proteomic profiling from 10 µL of plasma.

Main Results:

• Achieved plasma proteome coverage of 1136 proteins, a 49% increase over PCA alone and 2.6-fold over standard protocols.
• Significantly improved detection of low-abundance proteins, covering 101 FDA-approved circulating biomarkers.
• Maintained comprehensive metabolomic coverage (1174 metabolites) with superior reproducibility and consistency compared to standard methods.

Conclusions:

• The orthogonal extraction workflow enhances proteomic depth and preserves metabolomic integrity from microliter-scale plasma samples.
• This integrated approach facilitates protein-centric biomarker discovery with orthogonal metabolic insights.
• The workflow offers a transformative tool to accelerate the clinical translation of biomarkers by overcoming limitations of standalone proteomics.