Comparison of deep plasma proteome enrichment methods

Plasma is a complex biological fluid containing extracellular vesicles (EVs), residual platelets, and soluble proteins, all of which can serve as biomarkers for pathological conditions. While conventional plasma proteomics typically identifies hundreds of proteins, recent enrichment strategies have expanded coverage to thousands. It is not clear yet whether these methods enrich preferentially different classes of protein and whether they allow for reliable quantification Here, we compared three common advanced proteomic workflows—Seer Proteograph (nanoparticle-based), MagNET (SCX magnetic beads), and ENRICHplus (PreOmics)—as well as EV enrichment obtained by centrifugation. We aimed to explore the content in soluble proteins, EV cargo, and platelet-derived proteins in platelet-poor plasma (PPP) from healthy donors after the enrichments. Samples were analyzed using timsTOF platforms and quantified with DIA-NN in library-free mode. Functional insights were obtained using Over-Representation Analysis (ORA), Human Protein Atlas annotations, and EV-specific markers. Quantification was evaluated comparing each method to neat plasma using protein coefficient of variation and point-biserial correlation. We quantified an average of ~4500 proteins with EV centrifugation, ~4000 with Seer, ~2800 with ENRICHplus, ~2300 with MagNET, and ~900 with neat plasma. Each method enriched distinct protein signatures: EV markers such as CD81 were predominantly detected in EV preparations; lipoproteins were enriched in ENRICHplus; cytokine and hormone signaling pathways were most evident in Seer. Platelet protein intensity was directly correlated with total protein identifications but did not compromise quantification of low-abundance proteins. Across 50 healthy individuals, Proteograph consistently demonstrated reproducible enrichment and depletion patterns, with notable exceptions for proteins such as FCN3, F11, and FCGBP.