Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles

Abstract

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer‐modified (poly(DMA‐GMA‐MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System

“…Only low intense 'spiky' signals were registered in the latter case. In our previous works, these signals were assigned to the presence of macromolecular aggregates that are a result of MVs squeezing during ultracentrifugation [29,31]. Thus, it can be concluded that the concentration of fluorescent compounds, co-migrating with the main signal (Figure 4b), is relatively low in relation to MVs, and their detection is a result of significantly greater sensitivity of LIF detector.…”

“…The protein content of MVs samples was quantified by the bicinchoninic-acid assay (BCA) as described in [31]. In brief, the isolates were mixed with 6% SDS solution in a 9 to 1 volume ratio and incubated with BCA kit reagents according to the microplate protocol recommended by the vendor (ThermoFisher Scientific, Waltham, MA, USA).…”

“…Fluorescence was measured with a laser-induced fluorescence detector (LIF) using 488 nm laser for excitation and 520 nm filter for emission. A detailed description of the capillary conditioning procedures was described in [31].…”

Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis

“…These alterations were reported to affect the biological properties of EVs [27]. Our group has recently demonstrated that the CE can distinguish EVs from macromolecular aggregates that were formed during direct UC [17,18]. The aggregates were observed in electropherograms in a form of highly efficient (>1 mln plates m −1 ) signals, which in the literature are often referred to as spikes [28,29].…”

“…Our group explored the CE as a tool for bacterial EVs characterization. It was shown that the use of simple UV detection is sufficient for quantification of EVs and detection of macromolecular aggregates presence in isolates [17,18]. Morani and coworkers demonstrated the application of CE with laser-induced fluorescence detection (LIF) for the characterization of human and bovine vesicles stained with fluorescein derivative [19].…”