Extracellular vesicle (EV) characterization: Is gas-phase electrophoresis on a nES GEMMA instrumentation yielding valuable data? V. U. Weiss, Vienna/AUT, S. Steinberger, Vienna/AUT, V. Weber, Krems/AUT, M. Marchetti-Deschmann, Vienna/AUT Assoc. Prof. Dr. Victor U. Weiss, TU Wien, Getreidemarkt 9/164, 1060 Vienna/AUT Gas-phase electrophoresis applying a nano Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) instrument also known as nES Differential Mobility Analyzer (nES DMA) enables bionanoparticle characterization. Analytes are transferred from a volatile electrolyte solution to the gas-phase via an nES process followed by particle drying and charge-equilibration. As a result, mostly neutral yet also a considerable amount of single-charged, surface-dry particles is obtained. Furthermore, due to mild conditions during the nES process, even larger, non-covalently bound biological moieties remain in their native state. Subsequently, analytes are separated in the nano DMA part of the nES GEMMA by voltage scanning in a tunable electric field and an orthogonal high sheath flow of particle-free air. As particles are single-charged (neutral species are not regarded), separation of analytes is solely based on their dry particle electrophoretic mobility (EM) diameter. Furthermore, in good accordance with a recommendation of the European Commission for nanoparticle research (updated version 2022/C 229/01, June 10th, 2022), particle detection is based on the number of particles – analytes are counted after gas-phase electrophoretic separation yielding a corresponding spectrum comparing EM diameter and particle count values. [1] Such a setup enables detection of smaller sized analytes next to larger ones. In previous work, nES GEMMA has shown its applicability for the analysis of liposomes, vesicles consisting of a lipid bilayer and encapsulating an aqueous lumen. [2, 3] Gas-phase electrophoresis enabled the characterization of these vesicles in terms of size distribution, particle number concentration and the occurrence of smaller sized building blocks next to large vesicles. Also, offline hyphenation of gas-phase electrophoresis with orthogonal analysis methods, for instance atomic force microscopy (AFM) or mass spectrometry (MALDI MS) was shown. [4] In vivo, extracellular vesicles (EVs), being cell-derived and essential for cell/cell communication, are comparable to in vitro formed liposomes. EVs are envisioned as personalized pharmaceutical cargo transporters, with the goal to significantly reduce the side effects of the carrier. However, such an application underscores the need for their in-depth analytical characterization. In this context, we succeeded in porting gas-phase electrophoresis of liposomes to the characterization of EVs, demonstrating protein co-purification [5] and loss of EV stability upon further polishing of vesicle preparations [6]. Literature: [1] S. L. Kaufman, Anal. Chem., 1996, 68, 11. [2] V. U. Weiss, Analyst, 2016, 141, 21. [3] C. Urey, Int. J. Pharm., 2016, 513, 1-2. [4] V. Weiss, J. Pharm. Biomed. Anal., 2020, 179, 112998. [5] S. Steinberger, Anal. Bioanal. Chem., 2021, 413, 30. [6] S. Steinberger, Membranes, 2022, 12, 9.