Enriching extracellular vesicles for mass spectrometry

Abstract

Extracellular vesicles from plasma, other body fluids and cell culture media hold great promise in the search for biomarkers. Exosomes in particular, the vesicle type that is secreted after being produced in the endocytic pathway and having a diameter of 30–150 nm, are considered to be a conveyance for signaling molecules and, therefore, to hold valuable information regarding the health and activity status of the cells from which they are released. The vesicular nature of exosomes is central to all methods used to separate them from the highly abundant proteins in plasma and other fluids. The enrichment of the vesicles is essential for mass spectrometry‐based analysis as they represent only a very small component of all plasma proteins. The progression of isolation techniques for exosomes from ultracentrifugation through chromatographic separation using hydrophobic packing materials shows that effective enrichment is possible and that high throughput approaches to exosome enrichment are achievable.

Meta-analysis refinement of plasma extracellular vesicle composition identifies proplatelet basic protein as a signaling messenger in type 1 diabetes

“…Several analytical techniques have been developed for the isolation of EVs, including ultracentrifugation (UC), density gradient ultracentrifugation (DGUC), cushion ultracentrifugation (CUC), polymer-based precipitation (PP), size-exclusion chromatography (SEC) and immunocapture (IC) (Burton et al , 2021; Coumans et al , 2017). These techniques all have their advantages, but they also suffer from unique contaminant profiles (Burton et al ., 2021; Coumans et al ., 2017). For instance, UC co-precipitates particles of similar density while SEC co-fractionates particles of similar sizes (Burton et al ., 2021; Coumans et al ., 2017).…”

“…These techniques all have their advantages, but they also suffer from unique contaminant profiles (Burton et al ., 2021; Coumans et al ., 2017). For instance, UC co-precipitates particles of similar density while SEC co-fractionates particles of similar sizes (Burton et al ., 2021; Coumans et al ., 2017). Sequential purification steps have also been explored (Xu et al , 2015).…”

Pattern Analysis of Organellar Maps for Interpretation of Proteomic Data

“…Displaying vesicular protein abundance on the LOPIT plot allows patterns from disparate isolation techniques to be recognized [ 15 ]. The vesicular proteins from ultracentrifugation, size exclusion chromatography, and polymer-capillary channel hydrophilic interaction chromatography isolation from blood serum have a similar profile when displayed on a LOPIT plot.…”

“…Extracellular vesicles are important vehicles of intercellular communication that can potentially be used for disease identification, assessment of response to therapy, and for drug delivery [ 13 , 14 ]. Identifying low abundance proteins in the vesicle preparations via mass spectrometry has emerged as a powerful tool in biomarker discovery and in epidemiological studies [ 15 ]. It remains a challenge to enrich vesicles from plasma, urine, and other biological matrixes.…”

Extracellular vesicles in β cell biology: Role of lipids in vesicle biogenesis, cargo, and intercellular signaling

“…Polymer-based precipitation has the advantage that it is a rapid isolation technique allowing for isolation and characterization of large numbers of samples; however, polymers can alter the composition of the EVs, and the levels of polymers required to remove contaminating lipoproteins result in precipitation of a majority of the EVs of interest. Similarly, while size exclusion chromatography is gentle and has excellent recovery and repeatability/reproducibility, EVs share similar hydrodynamic diameters with large plasma lipoproteins, which results in co-fractionation of these distinct populations that can confound compositional analysis [ [152] , [153] , [154] , [155] ]. This issue remains an ongoing concern that receives continually refined recommendations by the International Society for Extracellular Vesicles [ 8 ].…”