Addressing MISEV guidance using targeted LC‐MS/MS: A method for the detection and quantification of extracellular vesicle‐enriched and contaminant protein markers from blood

Extracellular Vesicles

Extracellular vesicles (EVs) are membrane‐bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV‐associated markers and negative contaminant markers based on commonly co‐isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high‐throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC‐MS/MS) assay for the quantification of markers associated with EVs and non‐vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations.

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Recent Publications

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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