Characterization of extracellular vesicles via nES GEMMA, NTA and MS/MS regarding purity and vesicle integrity

Abstract

Characterization of extracellular vesicles via nES GEMMA, NTA and MS/MS regarding purity and vesicle integrity Stephanie Steinberger1, Sobha Karuthedom George2, Lucia Lauková2, René Weiss2, Carla Tripisciano2, Ruth Birner-Gruenberger1, Viktoria Weber2, Martina Marchetti-Deschmann1, Victor U. Weiss1 1 Institute of Chemical Technologies and Analytics, TU Wien, Vienna, Austria 2 Center for Biomedical Technology, Dept. Biomed. Res., University for Continuing Education Krems, Krems, Austria Email: stephanie.steinberger@tuwien.ac.at

Introduction Extracellular vesicles (EVs) are bio-nanoparticles shed by organisms, consisting of a lipid bilayer and an aqueous lumen. They are in the focus of medicinal research due to their significant role in cellular processes and their potential for biomarkers. However, their heterogeneity renders vesicle characterization challenging. In this context, EVs were analyzed via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES-GEMMA) [1], determining their electrophoretic mobility diameter (EMD, i.e., the surface-dry particle diameter) based on particle-number detection. Furthermore, nES-GEMMA enables the detection of smaller analytes next to larger ones.

Methods EVs from human blood were isolated via ultracentrifugation. The original sample buffer was exchanged to 40 mM ammonium acetate, pH 8.4 (NH4OAc), with centrifugal filters (10 kDa, MWCO, polyethersulfone). Analyses were performed on nanoparticle tracking analysis (NTA, Particle Metrix, Inning am Ammersee, Germany) and nES-GEMMA instrumentation (TSI Inc., Shoreview, MN, USA). Proteinaceous contaminants were characterized with SDS-PAGE and tryptic in-gel digest followed by MALDI TOF/RTOF MS (UltrafleXtreme, Bruker, Billerica, MA, USA) in positive mode. Contaminants were depleted via size exclusion chromatography (SEC). Samples prior and after SEC were analyzed via timsTOF MS/MS (Bruker).

Results Focusing on nES GEMMA, we exchanged the original EV sample buffer to NH4OAc. This led to a particle loss of 60-90% from 1011-1012 vesicles/mL starting value, as determined by NTA. Subsequently, nES GEMMA revealed a broad nanoparticle size distribution from 20 nm to 90 nm EMD. Concomitantly, mostly co-isolated proteins, not detectable via NTA, were detected in the lower EMD range. SDS PAGE and MALDI TOF/RTOF MS identified the main co-isolated proteins as hemoglobin, α-2-macroglobulin, and β-actin-(like)-protein. An additional SEC purification results in a significant depletion of co-isolated proteins. However, the overall EV hydrodynamic size distribution (NTA) and sample proteome (LC-ESI-MS/MS) were not affected. However, SEC contributes also to a further loss of vesicle numbers [2]. Furthermore, SEC samples showed signs of rupture and damage of vesicles upon nES GEMMA [3].

Innovative aspects • nES GEMMA based EV characterization concerning size and particle numbers • Detection of co-purified, proteinaceous contaminants via nES GEMMA • Influence of sample preparation on vesicle integrity can be assessed

Acknowledgements Project support by the NÖ Forschungs- und Bildungsges.m.b.H. (NFB, LSC16-018).

References [1] Kaufman, S. L. et al., Analytical Chemistry 1996, 68 (11), 1895-1904. [2] Steinberger, S. et al., Analytical and Bioanalytical Chemistry 2021, 413 (30), 7341-7352. [3] Steinberger, S. et al., Membranes 2022, 12 (9), 872.