Gas-phase electrophoresis (nES GEMMA instrumentation) in extracellular vesicle (EV) research

Abstract

Gas-phase electrophoresis (nES GEMMA instrumentation) in extracellular vesicle (EV) research Victor U. Weiss1) 1) TU Wien E-mail: victor.weiss[at]tuwien.ac.at Introduction: Gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyser (nES GEMMA) separates single-charged particles obtained after a nES process with subsequent surface-drying and charge equilibration. Separation is based on the electrophoretic mobility diameter (EMD) corresponding to the particle diameter for spherical analytes. By voltage variation, particles of different EMD successively reach the instruments’ detector unit yielding particle number concentrations in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011, updated in 2022). Materials and Methods: EVs from human blood were isolated via ultracentrifugation. Subsequently, the original sample buffer was exchanged to volatile ammonium acetate. This step was monitored via nanoparticle tracking analysis (NTA, Particle Metrix, Inning am Ammersee, Germany). Gas-phase electrophoresis was on a nESGEMMA instrumentation (TSI Inc., Shoreview, MN, USA). Results: and Discussion: In previous work, the applicability of nES GEMMA for the analysis of liposomes, vesicles consisting of a lipid bilayer and encapsulating an aqueous lumen, was demonstrated. nES GEMMA enabled the characterization of vesicles in terms of size distribution, particle number concentration and the occurrence of smaller sized building blocks. Also, offline hyphenation of gas-phase electrophoresis with orthogonal analysis methods was shown. In vivo, cell-derived EVs, being e.g. part of cell/cell communication, are comparable to liposomes. They are envisioned as pharmaceutical cargo transporters, explaining the need for their analytical characterization. We succeeded in porting gas-phase electrophoresis of liposomes to the characterization of EVs, e.g. demonstrating protein co-purification and loss of EV stability upon further polishing of vesicle preparations.