Oral Presentation Australasia Extracellular Vesicles Conference 2017

Optimisation of Extracellular Vesicle Purification from Serum and Plasma for Proteomics Analysis (#2)

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

Obtaining high purity and high yield samples for mass spectrometry analysis has presented a challenge for extracellular vesicle (EV) research. In particular, when working with highly complex fluids such as serum and plasma, the signal from high abundance species such as albumin and immunoglobulins can impede the detection of low abundance EV-associated species. Preparative methods such as density gradient ultracentrifugation have been used to reduce the presence of contaminant protein 1, however, such methods are laborious and very low throughput.  To this end, we aimed to develop a robust, high throughput method for preparing high quality EV isolates from serum or plasma suitable for proteomics analysis. We explored two techniques: size exclusion chromatography (SEC) and Proteinase K digest. Size exclusion chromatography has previously been employed for the purification of plasma EVs for proteomic analysis 2, however, to our knowledge a thorough optimisation of the SEC workflow for downstream mass spectrometry has not yet been performed. We performed this evaluation, and investigated the effect of several parameters including column bed volume, fraction volume and buffer composition on sample purity. Proteinase K is a serine protease which exhibits proteolytic activity across a wide range of conditions. We also investigated the feasibility of Proteinase K digestion coupled with ultrafiltration to deplete high abundance proteins from plasma and/or serum EV samples, whilst maintaining the integrity of EV-associated proteins. In total, we present a comprehensive analysis of Proteinase K and SEC -purified EVs, including mass spectrometry data as well detailed physical characterisation by tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA). We anticipate that these results will guide the development of a robust workflow to enable high quality proteomic characterisation of serum and/or plasma derived EVs.

  1. Kalra, H. et al. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics 13, 3354-3364, (2013).
  2. de Menezes-Neto, A. et al. Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals. Journal of Extracellular Vesicles 4, 27378, (2015).