An Orthogonal Extraction Workflow for High-Depth Plasma Proteomics with Metabolomic Compatibility from Low Microliter-Scale Samples

ABSTRACT: Herein, we report an orthogonal extraction workflow that achieves enhanced proteomic profiling depth while preserving metabolomic compatibility from a mere 10 µL of plasma. We demonstrate that a ternary solvent mixture (TSM) of methanol, acetonitrile, and acetone exhibits distinct chemical orthogonality to perchloric acid (PCA) precipitation, with each strategy recovering reproducible yet partially non-overlapping subsets of plasma proteins. By combining orthogonal extraction with PCA and TSM to deplete high-abundant proteins, we expanded plasma proteome coverage to 1136 proteins—representing a 49% increase compared to PCA alone and a 2.6-fold enhancement relative to the standard neat plasma processing protocol. This marked improvement in low-abundance protein detection enabled efficient coverage of 101 FDA-approved circulating biomarkers. For metabolomics, the workflow retained comprehensive coverage (1174 metabolites) comparable to standard methanol extraction, with superior qualitative reproducibility and quantitative consistency across technical replicates. This workflow may enable protein-centric biomarker discovery augmented by orthogonal metabolic insights from microliter-scale samples, providing a transformative tool to surmount the limitations of standalone proteomics and accelerate the clinical translation of candidate biomarkers.

ORGANISM(S): Homo Sapiens

SUBMITTER: Weijie Qin  

PROVIDER: PXD073964 | iProX | Tue Feb 03 00:00:00 GMT 2026

REPOSITORIES: iProX