Combining Anion Exchange and Size Exclusion Chromatography for Extracellular Vesicle Enrichment from Small Volumes of Human and Mouse Plasma for Quantitative Proteomics

Abstract Extracellular vesicles (EVs) are promising biomarkers, yet their proteomic analysis from plasma is hampered by low abundance and co-purification of contaminants (e.g., lipoproteins, platelets) and technical variability, particularly in small-volume animal models. We developed and validated a modular protocol integrating Size Exclusion Chromatography (SEC) with Strong Anion Exchange (SEC-SAX) specifically tailored for quantitative LC-MS proteomics from small starting volumes (150 μl of plasma). SEC alone successfully removed 99% of Albumin, and the SAX step significantly enriched EVs over contaminating lipoproteins. Downstream single pot solid phase enhanced (SP3) sample prep and STAGE tip solid phase extraction ensured maximum proteome depth. Critical confounding factors were objectively assessed: Platelet Factor 4 (PF4) was confirmed as a highly sensitive platelet marker, confirming the necessity of meticulous plasma preparation. Sample hemolysis impacted the plasma EV proteome data. As such, an objective measure (nanodrop spectrophotometer) of hemolysis and exclusion of hemolysed samples (heme >0.3 mg/ml) is recommended. The protocol is applicable to both human and mouse plasma as demonstrated by EV enrichment and quantification of biomarker proteins associated with neurodegenerative diseases from eight individual mouse plasma samples. Manuscript Highlights Developmental workflow for a quantitative SEC-SAX protocol for EV proteomics from small plasma volumes (150 μl). A range of variables tested including SAX beads amount, digestion buffer, digestion time, STAGE tip solid phase extraction, SAX elution buffer and sample filtration. The SAX step significantly enhances EV proteome depth by increasing EV purity in relation to ApoB lipoproteins. Shows the impact of the major confounding factors of sample hemolysis and platelet contamination on the EV proteome. Platelet contamination increases the number and abundance of proteins detected including known disease biomarkers and sample hemolysis is associated with proteins derived from platelet and red blood cell derived EVs. Platelet Factor 4 (PF4) is identified and confirmed as a sensitive marker for platelet contamination. Applicable to both human and mouse plasma. Competing Interest Statement The authors have declared no competing interest.