A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

Abstract

Proteomic analysis of exosomes (EX) poses a significant challenge. A ‘gold-standard’ method for plasma EX enrichment for downstream proteomic analysis is yet to be established. Our group has performed a comprehensive study of multi-dimensional enrichment methods to determine their efficiency for protein isolation. Methods were evaluated for their capacity to a) successfully isolate and enrich EX from blood plasma, b) minimise the presence of highly abundant plasma proteins, and c) result in the optimum representation of EX proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS). Blood plasma from four animals (Bos taurus) of similar physical attributes and genetics were used. Three methods of EX enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These enrichment methods were combined to create four groups for methodological evaluation: UC+SEC, UC+SEC+UF, SEC+UC and SEC+UF. UC+SEC yielded the highest number of protein IDs. Plasma protein identification was the least in SEC+UC, but this method yielded the lowest number of protein IDs overall. UC+SEC+UF decreased EX protein ID and did not improve purity compared to UC+SEC. Our data suggest that the method and sequence of EX enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

Differentially Expressed Extracellular Vesicle, Exosome and Non-Exosome miRNA Profile in High and Low Tick-Resistant Beef Cattle

“…Additionally, the 100,000 rcf UC pellet may contain circulating miRNA and other RNA fragments. A recent study illustrates the presence of higher number of EX proteins using the UC+SEC isolation and enrichment strategy for blood plasma EX (Turner et al, 2021). UC followed by SEC of plasma samples may therefore contain uniquely sorted exosomal cargo and provides a better crosssection of genetic materials including miRNA.…”