Name: Extracellular Vesicle (EV) Isolation from Human Urine Samples Using the EVtrap Approach
Uploaded: 2023-10-27T13:10:00
Description: Watch this Scientific Journal Video about Extracellular Vesicle EV Isolation from Human Urine Samples Using the EVtrap Approach at JoVE.com

extracellular vesicle (ev) isolation from human urine samples using the evtrap approach

सटीक बायोफ्लुइड विश्लेषण के लिए ईवी अलगाव को बढ़ाना

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles secreted by all types of cells and are present in biofluids such as blood, urine, saliva, etc.1,2,3,4. EVs carry a cargo of diverse bioactive molecules which reflect the physiological and pathological state of their host cells and, therefore function as crucial factors in disease progression4,5,6. Moreover, extensive studies have established that EV-based disease markers can be identified prior to the onset of symptoms or the physiological detection of tumors5,6,7.
Phosphorylation acts as a key mechanism in cellular signaling and regulation. Therefore, phosphoproteins provide a valuable source for biomarker discovery as aberrant phosphorylation events are associated with dysregulated cellular signaling pathways and metastatic disease development such as cancer8,9,10. Although profiling phosphorylation dynamics allows for the identification of disease-specific phosphoprotein signatures as potential biomarkers, the low abundance and dynamic nature of phosphoproteins pose major challenges in developing phosphoproteins as biomarkers11,12. Notably, the low-abundant phosphoproteins encapsulated within EVs are protected from external enzymatic digestion in the extracellular environment8. Consequently, EVs and EV-derived phosphoproteins offer an ideal source for biomarker discovery in the early-stage detection of cancer and other diseases.
Although analysis of protein phosphorylation in EVs offers a valuable resource for understanding cancer signaling and early-stage disease diagnosis, the lack of efficient EV isolation methods presents a major barrier. EV isolation is commonly achieved through differential ultracentrifugation (DUC)13. However, this method is time-consuming and is not suitable for clinical implications due to low throughput and poor reproducibility13,14. Alternative EV isolation approaches, such as polymer-induced precipitation15, are limited by low specificity due to co-precipitation of non-EV proteins. Affinity-based approaches, including antibody-based affinity capture16 and affinity filtration17, offer enhanced specificity but are restricted to a relatively low recovery rate due to small volume.
To address the issues in exploring phosphoprotein dynamics in EVs, our group has developed extracellular vesicles total recovery and purification (EVtrap) technique based on chemical affinity to capture EVs onto functionalized magnetic beads18. Previous results have demonstrated that this magnetic bead-based EV isolation method is highly effective in isolating EVs from a wide range of biofluid samples and is able to achieve much higher EV yield while minimizing contamination compared to DUC and other existing isolation methods18,19. We have successfully utilized EVtrap and a titanium-based phosphopeptide enrichment method developed by our group20 to profile the phosphoproteome of EVs derived from diverse biofluids and to detect potential phosphoprotein biomarkers for various diseases19,21,22.
Here, we present a protocol based on EVtrap for the isolation of circulating EVs. The protocol focuses on the urinary EVs. We also demonstrate the characterization of isolated EVs using western blotting. We then detail the sample preparation and mass spectrometry (MS) acquisition for both proteomics and phosphoproteomics analyses. This protocol provides an efficient and reproducible workflow for profiling the urinary EV proteome and phosphoproteome, which will facilitate further studies on EVs and their clinical applications23.

लेखक स्पॉटलाइट: रोग बायोमार्कर डिस्कवरी में उच्च-थ्रूपुट प्रोटिओमिक्स के लिए ईवीट्रैप को आगे बढ़ाना 

प्रोटिओमिक्स और फॉस्फोप्रोटेओमिक्स विश्लेषण के लिए बायोफ्लुइड से बाह्य पुटिकाओं का रासायनिक आत्मीयता-आधारित अलगाव

We are working on extracellular vesicle-based biomarker discovery. One important step is to isolate them efficiently and selectively. EVtrap, which is based on magnetic bead-based separation, can really help us by providing high throughput, easy to use, in particular, easy for clinical applications.We have been using this protocol for discovery in proteins and the phosphoprotein biomarkers for different diseases such as breast cancer, kidney cancers, and Parkinson's Disease. This biomarker is derived from biofluid. EVs can potentially be used for liquid biopsies, which can further improve clinical diagnosis and the treatment.Currently, the most commonly used approach for extracellular vesicle isolation are ultracentrifugation and precipitation, but those methods are limited to co-isolation of contaminants and also low efficiency. So our EVtrap method will provide a more effective way for isolating EVs, which will allow for a more reproducible and sensitive method and also will benefit downstream analysis. We are working on two things.First, we are continue developing the methods and protocols for EV isolation and a downstream analysis, in particular, a mass backup-based analysis. Second one is we are exploring different application in clinical field because of the advantage by the EVtrap.

Watch this Scientific Journal Video about Extracellular Vesicle EV Isolation from Human Urine Samples Using the EVtrap Approach at JoVE.com

Extracellular Vesicle (EV) Isolation from Human Urine Samples Using the EVtrap Approach  

एक्स्ट्रासेलुलर वेसिकल (EV) EVtrap दृष्टिकोण का उपयोग करके मानव मूत्र के नमूनों से अलगाव

शुरू करने के लिए, स्वस्थ व्यक्तियों से मूत्र के नमूने एकत्र करें। बड़े एपोप्टोटिक निकायों में सेल मलबे को खत्म करने के लिए 15 मिलीलीटर शंक्वाकार ट्यूब में 12 मिलीलीटर नमूना अपकेंद्रित्र। फिर, सतह पर तैरनेवाला के 10 मिलीलीटर एक ताजा 15 मिलीलीटर ट्यूब में हस्तांतरण.नमूने में लोडिंग बफर का एक मिलीलीटर और ईवी ट्रैप बीड स्लरी के 200 माइक्रोलीटर जोड़ें। अंत रोटेशन पर अंत के साथ 30 मिनट के लिए कमरे के तापमान पर नमूना सेते हैं. नमूना गोली करने के लिए, एक चुंबकीय विभाजक रैक पर 15 मिलीलीटर शंक्वाकार ट्यूब रखें।ध्यान से सतह पर तैरनेवाला हटा दें और धोने बफर के एक मिलीलीटर में मोती फिर से निलंबित. फिर, निलंबन को 1.5 मिलीलीटर माइक्रो सेंट्रीफ्यूज ट्यूब में स्थानांतरित करें और धीरे से पिपेट को बाह्य पुटिका या ईवी-बाध्य मोतियों को फिर से निलंबित करने के लिए। 1.5 मिलीलीटर ट्यूबों के लिए एक चुंबकीय विभाजक रैक पर माइक्रो अपकेंद्रित्र ट्यूब रखें।एक P200 विंदुक का प्रयोग, ध्यान से सतह पर तैरनेवाला महाप्राण. अब, धोने बफर के एक मिलीलीटर के साथ मोती धो लें, कमरे के तापमान पर पीबीएस के एक मिलीलीटर के साथ दो washes के बाद. पांच मिनट के लिए ताजा तैयार 100 मिलीमोलर Triethanolamine के 100 microliters के साथ मोती सेते हैं.1.5 मिलीलीटर माइक्रो अपकेंद्रित्र ट्यूब चुंबकीय विभाजक रैक का उपयोग करके, ईवीएस युक्त विलुप्त समाधान एकत्र करें। eluded समाधान के संयोजन के बाद, चार डिग्री सेल्सियस पर एक वैक्यूम अपकेंद्रित्र सांद्रक का उपयोग कर, eluit सूखी.

Research

JoVE Journal

Biochemistry

Extracellular Vesicle (EV) Isolation from Human Urine Samples Using the EVtrap Approach

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...