Reham Mohamed Ali Marzouk (Chungnam National University)

Reham Mohamed Ali Marzouk (Chungnam National University)

Reham Mohamed Ali Marzouk (Chungnam National University)

Jeongkwon Kim (Chungnam National University)

Exosomes are nanosized extracellular vesicles, typically measuring between 30 and 120 nm. Their potential applications in disease diagnostics, therapeutics, and drug delivery have recently attracted considerable attention. This study presents a method for enriching and isolating serum exosomes using a 10,000 polyethylene glycol (PEG) precipitation solution. We also compared this PEG solution with the commercially available ExoQuick kit. To remove cells and larger molecules, the serum was subjected to two rounds of low-speed centrifugation. The supernatant was then filtered, mixed with either PEG or ExoQuick, incubated at 4°C, and centrifuged at 1,500 × g. The resulting pellet was resuspended in Phosphate-Buffered Saline (PBS), with the process repeated four times. Size exclusion chromatography confirmed the successful enrichment of exosomes from the serum samples. Western blot analysis and scanning electron microscopy provided further validation. Following the isolation of exosomes from serum using both approaches, the exosomes were subsequently lysed and subjected to proteolytic digestion. The resulting peptides were then analyzed to profile the exosomal protein content using nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Our results indicate that the PEG precipitation method is highly efficient and performs similarly to the more expensive ExoQuick kit, offering a cost-effective and efficient alternative for exosome enrichment from blood serum.