Oral Presentation Australasian Extracellular Vesicles Conference 2018

Optimisation of size exclusion chromatography for enrichment and proteomics analysis of extracellular vesicles from clinically relevant samples (#4)

Rebecca E Lane 1 , Darren Korbie 1 , Matt Trau 1 2 , Michelle M Hill 3 4
  1. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia
  3. QIMR Berghofer Medical Research Institute, Herston, QLD, Australia
  4. The University of Queensland Diamantina Institute, Translational Research Institute, Woolloongabba, QLD, Australia

Proteomics analysis of EVs from plasma and serum samples is challenging due to the large abundance of serum and plasma proteins which impede detection of the EV proteome. Size Exclusion Chromatography (SEC) has been proposed as a preparative method to enrich EVs from a variety of biological fluids and deplete highly abundant contaminant protein. In this investigation, we demonstrate that an optimised SEC workflow enables rapid EV enrichment from low volumes (<500µL) of clinically relevant plasma samples. After testing a variety of SEC configurations, it was determined that a 10 mL Sepharose 4B column in PBS provided maximum enrichment of EVs. The optimised method was then used to enrich EVs from healthy plasma samples spiked with approximately 1%, 5% and 10% MDA-MB-231 breast cancer cell derived- EVs along with cell line EVs alone. NanoLC-MS/MS analysis of the enriched EVs indicated that cancer EV-associated proteins could be identified from as little as 1% spiked EV content in healthy plasma. To our knowledge, this is the first attempt to quantify the limit of detection of disease associated EVs in this manner. Our method was subsequently applied to detect disease-relevant proteins from the plasma of a cohort of triple positive breast cancer patients (n=10) and healthy controls (n=10).  In total, we demonstrate that SEC coupled to nanoLC-MS/MS enables detection of relevant biomarkers from small volumes of plasma and/or serum.  This method has the potential to be used for biomarker discovery and/or validation across clinical sample cohorts.