Name: Preparation of Dried Extracellular Vesicle (EV) Sample for Proteomics and Phosphoproteomics Analysis
Uploaded: 2023-10-27T13:10:00
Description: Watch this Scientific Journal Video about Preparation of Dried Extracellular Vesicle EV Sample for Proteomics and Phosphoproteomics Analysis at JoVE.com

preparation of dried extracellular vesicle (ev) sample for proteomics and phosphoproteomics analysis

Miglioramento dell'isolamento delle vescicole extracellulari per un'analisi precisa dei biofluidi

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.

Autore in primo piano: Avanzamento di EVtrap per la proteomica ad alto rendimento nella scoperta di biomarcatori di malattia 

Isolamento chimico basato sull'affinità di vescicole extracellulari da biofluidi per analisi proteomiche e fosfoproteomiche

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 Preparation of Dried Extracellular Vesicle EV Sample for Proteomics and Phosphoproteomics Analysis at JoVE.com

Preparation of Dried Extracellular Vesicle (EV) Sample for Proteomics and Phosphoproteomics Analysis  

Preparazione di un campione di vescicole extracellulari essiccate (EV) per l'analisi proteomica e fosfoproteomica

Per iniziare, prelevare il campione di vescicola extracellulare essiccata ottenuto dall'urina umana utilizzando l'approccio della trappola EV. Preparare un nuovo tampone di lisi con i componenti mostrati. Quindi aggiungere 100 microlitri di tampone di lisi per solubilizzare il campione EV essiccato.Riscaldare il campione a 95 gradi Celsius per 10 minuti agitando a 1.100 giri/min. Dopo aver raffreddato il campione a temperatura ambiente, diluirlo quintuplicando aggiungendo 400 microlitri di TEAB 50 millimolari. Misurare la concentrazione proteica utilizzando un kit di test BCA secondo le istruzioni del produttore.Aggiungere la miscela di tripsina e lisina C al campione e incubare a 37 gradi Celsius per una notte con agitazione a 1.100 giri/min. Quindi aggiungere 50 microlitri di acido trifluoroacetico al 10% o TFA per acidificare il campione. Inoltre, aggiungere 600 microlitri di acetato di etile ai campioni e vorticare la miscela per due minuti.Centrifugare il campione per tre minuti a 20.000 G e rimuovere con cautela lo strato superiore senza disturbare l'interfaccia. Essiccare la fase acquosa utilizzando un concentratore centrifugo sottovuoto. Ora risospendere il campione essiccato in 200 microlitri di TFA allo 0,1% per acidificare i peptidi.Per dissalare il campione, utilizzare un puntale di dissalazione CA18 e condizionarlo con 200 microlitri di TFA allo 0,1% e acetonitrile all'80%, seguiti da due lavaggi con 200 microlitri di TFA allo 0,1%. Caricare il campione di peptide acidificato nella punta e lavarlo tre volte con 200 microlitri di TFA allo 0,1%. Eluire i peptidi con 200 microlitri di TFA allo 0,1% e acetonitrile all'80%.Dopo aver prelevato l'eluente come dimostrato, risospendere il campione essiccato in fosfoproteomica in 200 microlitri di tampone di carico per l'arricchimento di fosfopeptidi. Aggiungere 50 microlitri di microlitri di microsfere al campione e agitare energicamente per 20 minuti a temperatura ambiente. Caricare il campione con le perle nella punta fritta e centrifugare per un minuto a 100 G.Lavare la punta successivamente con 200 microlitri di tampone di carico, lavando i tamponi uno e due.Posiziona la punta con le perline in un nuovo tubo per raccogliere i fosfopeptidi elusi. Aggiungere 50 microlitri di tampone di eluizione al puntale e centrifugare per due minuti a 20 G. Quindi asciugare i fosfopeptidi elusi utilizzando un concentratore per centrifuga sottovuoto.

Research

JoVE Journal

Biochemistry

Preparation of Dried Extracellular Vesicle (EV) Sample for Proteomics and Phosphoproteomics Analysis

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 ...

Ricerca

Biochimica

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

Isolamento delle vescicole extracellulari (EV) da campioni di urina umana utilizzando l'approccio EVtrap

Proteome/Phosphoproteome Analysis of the Extracellular Vesicle (EV) Samples Using LC-MS/MS

Analisi del proteoma/fosfoproteoma dei campioni di vescicole extracellulari (EV) mediante LC-MS/MS