Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma‐derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21‐gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at −80°, using a single freeze–thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle‐tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label‐free and gel‐free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular‐related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

“…[35] In this context, we would like to place special emphasis on platelet-derived EVs, which are the 70-90% of total circulating MVs in blood plasma. [36] In that case, many plateletderived MVs markers such as CD41, integrin β3, and CD61 were found in the label-free LC-MS/MS analysis, pointing a high enrichment of platelet-derived MVs in our samples. Regarding exosomes, we also found some markers such as CD9, HS70, and HSPA8.…”

“…In addition, we would like to underline that we found some EVs‐related markers (transmembrane and lipid‐bound proteins) for exosomes and MVs demonstrating the quality control of EVs isolation . In this context, we would like to place special emphasis on platelet‐derived EVs, which are the 70–90% of total circulating MVs in blood plasma . In that case, many platelet‐derived MVs markers such as CD41, integrin β3, and CD61 were found in the label‐free LC–MS/MS analysis, pointing a high enrichment of platelet‐derived MVs in our samples.…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…On the other hand, top-down proteomics has been less applied in EV research, mostly in exosome studies (e.g., [ 120 ]), even if it allows sequencing of intact proteins and their proteoforms without the need of proteolytic digestion before MS measurement. Indeed, despite the improvements in top-down proteomics, the high costs of the instrumentation, the low-efficiency of the dissociation techniques, and the complicated mass spectra lead to some limitations in the application of top-down proteomics to study specific modifications of intact circulating EVs proteins and characterize them [ 121 ].…”

Extracellular vesicles in patients in the acute phase of psychosis and after clinical improvement: an explorative study

“…Selection of anti-coagulant, concentration steps and freezing and thawing EV samples are all steps that may affect the size or concentration of vesicles in the samples (Barrachina et al, 2019). We aimed to start the EV isolation with fresh samples.…”