Method Evaluation of Liquid Biopsy Proteomics for Limited Plasma Volume

One of the biggest challenges in plasma proteomics is to reduce the dynamic range of plasma proteins to capture the expression of low-abundance proteins. Although abundant protein depletion and proximity extension assay are widely used for human samples, the application to animal species in non-clinical assessment remains limited. Here, we evaluated the Mag-Net and ENRICH-iST methods using 20 μl of plasma from both healthy and type 2 diabetes model rats. By combining with the optimized LC-MS method, Mag-Net identified more proteins than ENRICH-iST with high precision, although both methods identified over 5000 proteins. Using integrative proteomics and analytical approaches, including extracellular vesicle (EV) database comparison, Gene Ontology analysis, Simple Western, nanoparticle tracking analysis, and contamination assessments, we demonstrated in rat plasma that Mag-Net preferentially enriches EV proteins, whereas ENRICH-iST more evenly captures low-abundance proteins, including EV proteins. Regardless of the protein enrichment method employed, more than half of the tissue-specific proteins were classified as being specific to either the liver or the brain tissues. Whereas, several liver-specific proteins involved in protein-protein interactions, including Proz/Serpina10 and Ambp/Itih1/Itih2, were identified as exhibiting over a ten-fold increase in mean quantification values in the Mag-Net method compared to ENRICH-iST, suggesting enhanced enrichment of protein-protein interaction network. Ingenuity Pathway Analysis identified 219 downregulated and 80 upregulated proteins in the Mag-Net method dataset (fold change ≥ 1.5, p_BH ≤ 0.05), and 68 downregulated and 43 upregulated proteins in the ENRICH-iST method dataset (fold change ≥ 1.5, p_BH ≤ 0.2). Some significantly downregulated pathways, such as elastic fiber formation, were uniquely identified by the Mag-Net method. This approach was shown to be a powerful tool for investigating deregulated proteins and pathways. Potential applications include biomarker discovery and the assessment of drug efficacy and safety during development.