Method for isolation of small extracellular vesicles from different biofluids and workflow for Mass Spectrometry based-shotgun proteomics and RNA isolation

Small extracellular vesicles (sEVs) or exosomes are small-sized (30-150 nm), nanoparticles that are released from almost all cells under normal and pathophysiological conditions. The sEVs have a vital role in biological systems as they communicate and transfer their contents, such as proteins, lipids, and nucleic acids, from the cells of origin to nearby or distant cells. In recent years, there has been a growing interest in isolating sEVs for use in disease mechanisms, clinical diagnoses, and therapeutics. Due to their small size sEVs can be observed using electron microscopy. The size distribution and concentration were checked by Nanoparticle Tracking Analysis. Western blotting confirmed the presence of exosome markers. The ease of obtaining patient samples from biofluids like plasma, saliva, and urine makes them a valuable source for diagnostic purposes by isolating sEVs to diagnose and predict diseases early. However, there is no specific protocol to perform it altogether. We have developed an improved ultracentrifugation method using gradient ultracentrifugation and ultrafiltration, which results in higher sEVs purity and yield. We have tested this method on plasma, saliva, and urine at a single platform, and we have isolated proteins and RNA from exosomes for their downstream applications. Our method is simple to use and can be utilized for clinical research biomarker applications, in understanding disease mechanisms and monitoring its progressions from biofluid sample collections.