Extracellular vesicle characterization via nano-electrospray gas-phase electrophoretic mobility analysis (nES GEMMA)

Abstract

Extracellular vesicles (EVs) are emerging as future therapeutics and clinical diagnostic tools indicating a human’s health status. They are formed and released by cells into their surroundings for e.g., cell-to-cell communication, blood coagulation or even tumor metastasis. However, their heterogeneity and diversity in size and function makes their isolation and characterization difficult. For future medical applications purity of EV-containing samples is of importance. In this context, samples were analyzed via nano electrospray gas-phase electrophoretic mobility analysis (nES GEMMA) separating single-charged (bio)nanoparticles in the gas-phase at ambient pressure according to electrophoretic mobility (EM) diameters. nES GEMMA proved to be a reliable characterization technique for liposomes, strongly resembling EVs in constitution. In comparison to prevalent techniques like nanoparticle tracking analysis (NTA), determining the hydrodynamic particle diameter, nES GEMMA allows the determination of the surface dry particle diameter with particle-number based detection. Hence, larger molecules can be monitored next to smaller ones, independent of the chemical composition of an analyte. For nES GEMMA the EV sample buffer PBS was exchanged to a volatile ammonium acetate, necessary for nES-based measurements. nES GEMMA of EV-containing samples displayed a broad particle distribution, starting around 20 nm and tailing off to larger EM diameter values. Impurities, mostly co-isolated proteins, were detected in the lower EM diameter range (<20 nm). NTA corroborated the presence of vesicles and their loss during sample preparation in samples but failed to report those impurities despite high particle counts in nES GEMMA. For complete analysis these components were identified by mass spectrometry as hemoglobin, α-2-macroglobulin, and β-actin, correlating with the size/molecular mass calculations based on nES GEMMA (Steinberger et al., 2021, Anal. Bioanal. Chem.). An additional size exclusion chromatography (SEC) step after EV isolation depleted the mentioned impurities, evident in nES GEMMA spectra. The overall EV hydrodynamic size distribution and sample proteome was not influenced by SEC, verified by NTA and MS analysis. However, SEC contributes to a vesicle number loss and leads to an increased number of spikes in the spectra due to lipid fragments, indicating rupture and damage to vesicles. In conclusion, nES GEMMA is suitable for EV characterization and purity assessment, revealing co-isolates proteins not detectable via NTA. Additional SEC improves sample purity at a loss of vesicle numbers. This work was supported by the NÖ Forschungs- und Bildungsges.m.b.H (NFB), grant LSC16-018.