Multi-omic analysis of plasma-derived extracellular vesicles in breast cancer

Liquid biopsy is an emerging technology with potential to identify disease markers in patient breast cancer blood. Plasma contains abundant, biological nanoparticles called small extracellular vesicles (sEVs) which carry glycoconjugates (glycoproteins, glycolipids and proteoglycans). These glycoconjugates have been previously shown to change in breast cancer tissues and cell lines, making sEVs an appropriate liquid biopsy target to monitor glycosylation changes during disease and treatment. This thesis aimed to optimise a workflow for the multi-omic (proteomics and glycomics) analysis of plasma-derived sEVs from healthy individuals and breast cancer patient (before/after surgery). Three common isolation methods (ExoQuick, ultracentrifugation and size exclusion chromatography (SEC)) were compared to determine which method provides an enriched sEV population with minimal plasma protein contamination. The purity of isolated sEVs was assessed using nanoparticle tracking analysis and ‘omics analyses. The proteomics and Gene Ontology analysis of de-N-glycosylated peptides indicated that SEC-isolated sEVs contained the least non-sEV protein contamination, while glycomics analysis of N-glycans from sEV glycoproteins indicated that sEVs isolated using ExoQuick and ultracentrifugation showed great similarity to whole plasma glycosylation, indicating a high degree of plasma protein contamination compared with SEC. These findings form the basis for the further development of a multi-omic sEV analysis platform.