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

تعزيز عزل EV لتحليل دقيق للسوائل الحيوية

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.

تسليط الضوء على المؤلف: تطوير EVtrap للبروتينات عالية الإنتاجية في اكتشاف العلامات الحيوية للأمراض 

العزلة القائمة على التقارب الكيميائي للحويصلات خارج الخلية من السوائل الحيوية لتحليل البروتيوميات وعلم الفوسفوبروتيوميكس

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  

عزل الحويصلة خارج الخلية (EV) من عينات البول البشري باستخدام نهج EVtrap

للبدء ، اجمع عينات البول من الأفراد الأصحاء. جهاز طرد مركزي 12 ملليلتر من العينة في أنبوب مخروطي 15 ملليلتر للتخلص من حطام الخلايا في الأجسام المبرمج الكبيرة. ثم انقل 10 ملليلتر من المادة الطافية إلى أنبوب جديد سعة 15 ملليلتر.أضف ملليلتر واحد من مخزن التحميل المؤقت و 200 ميكرولتر من ملاط حبة مصيدة EV إلى العينة. احتضان العينة في درجة حرارة الغرفة لمدة 30 دقيقة مع الدوران من طرف إلى طرف. لحبيبات العينة, ضع الأنبوب المخروطي 15 ملليلتر على رف فاصل مغناطيسي.قم بإزالة المادة الطافية بعناية وأعد تعليق الخرز في ملليلتر واحد من المخزن المؤقت للغسيل. بعد ذلك ، انقل التعليق إلى أنبوب طرد مركزي صغير سعة 1.5 ملليلتر وماصة برفق لإعادة تعليق الحويصلة خارج الخلية أو الخرز المرتبط ب EV. ضع أنبوب الطرد المركزي الصغير على رف فاصل مغناطيسي لأنابيب 1.5 ملليلتر.باستخدام ماصة P200 ، استنشق بعناية طاف طفا. الآن ، اغسل الخرز بملليلتر واحد من المخزن المؤقت للغسيل ، متبوعا بغسلتين مع ملليلتر واحد من PBS في درجة حرارة الغرفة. احتضان الخرز مع 100 ميكرولتر من 100 ملليمولار ثلاثي إيثانولامين طازج لمدة خمس دقائق.باستخدام رف فاصل مغناطيسي لأنبوب الطرد المركزي الصغير سعة 1.5 ملليلتر ، اجمع الحل المراوغ الذي يحتوي على EVs. بعد الجمع بين المحاليل المستعصية ، جفف eluit ، باستخدام مكثف طرد مركزي فراغي عند أربع درجات مئوية.

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 ...