Abstract

Extracellular vesicles (EVs) are small, spherical particles produced by eukaryotic and prokaryotic cells, surrounded by a bilayer membrane and carrying various bioactive molecules, such as proteins, surface receptors, membrane and soluble proteins, lipids, and nucleic acids. EVs are of substantial interest because of their important roles in cell communication, epigenetic regulation and possible application in disease diagnosis and therapeutic strategies. Lipids are essential components of EVs since they are the building blocks of their bilayer membrane. Although the EV's lipid composition and its biological role are still unknown, this topic is gaining considerable attention in recent years. Lipidomics is a useful tool for characterisation of EVs' lipid profiles. However, such analysis can be challenging due to the limited size of sample (especially for EV obtained from primary cells), low lipid concentration and detection limits. Usually, extraction is performed before MS analysis to isolate lipids and remove interfering compounds such as proteins. In this work, we have tested the approach of direct injection LC-MS (DI-LC-MS) analysis of EV to characterize the lipidome without lipid extraction. RP-LC-Q-TOF-MS was used for the lipidomic profiling. EVs from various origins isolated by ultracentrifugation have been tested: from bovine and human sera, and EV derived from cultured eukaryotic and bacteria cells (incl. exosome-enriched small EVs, microvesicles and apoptotic bodies). We have evaluated the lipidome coverage and repeatability of the DI approach and compared it with the conventional extraction techniques used for the EVs' lipidomics. The results showed that injection of intact EVs allows the lipid analysis without a priori extraction, where high content of organic solvents in the mobile phase enables EV disintegration and lipid separation on the chromatographic column. The lipidome coverage obtained from conventional extraction and direct injection was comparable. However, the chemical background was reduced in DI approach in comparison to lipid extraction. The DI approach requires small injection volume (e.g. 0.1 µL depending on the EV particle number) and does not require an evaporation step. DI approach simplifies the procedure and makes EV lipidomic analysis fast and convenient without loss of lipidome coverage or repeatability.

Authors (6)