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

Forbedret EV-isolasjon for presis biofluidanalyse

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.

Forfatter i søkelyset: Fremme EVtrap for proteomikk med høy gjennomstrømning i oppdagelse av sykdomsbiomarkører 

Kjemisk affinitetsbasert isolering av ekstracellulære vesikler fra biofluider for proteomikk og fosfoproteomikkanalyse

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  

Fremstilling av tørket ekstracellulær vesikkel (EV) prøve for proteomikk og fosfoproteomikkanalyse

For å begynne, ta den tørkede ekstracellulære vesikkelprøven oppnådd fra den menneskelige urinen ved hjelp av EV-felle-tilnærmingen. Forbered en ny lysisbuffer med de viste komponentene. Deretter tilsettes 100 mikroliter lysisbuffer for å solubilisere den tørkede EV-prøven.Varm prøven ved 95 grader Celsius i 10 minutter mens du rister ved 1.100 RPM. Etter avkjøling av prøven til romtemperatur, fortynn den fem ganger ved å tilsette 400 mikroliter 50 millimolar TEAB. Mål proteinkonsentrasjonen ved hjelp av et BCA-analysesett i henhold til produsentens instruksjoner.Tilsett trypsin og lysin C-blanding til prøven en inkubere ved 37 grader celsius over natten med risting ved 1.100 RPM. Deretter tilsettes 50 mikroliter 10% trifluoreddiksyre eller TFA for å surgjøre prøven. Videre tilsettes 600 mikroliter etylacetat til prøvene og virvler blandingen i to minutter.Sentrifuge prøven i tre minutter ved 20.000 g og fjern forsiktig det øvre laget uten å forstyrre grensesnittet. Tørk den vandige fasen ved hjelp av en vakuumsentrifugekonsentrator. Nå resuspenderes den tørkede prøven i 200 mikroliter 0,1% TFA for å surgjøre peptider.For å avsalte prøven, bruk en CA18 avsaltingsspiss og kondisjoner den med 200 mikroliter 0,1% TFA og 80% acetonitril, etterfulgt av to vasker med 200 mikroliter 0,1% TFA. Legg den surgjorte peptidprøven i spissen og vask den tre ganger med 200 mikroliter 0,1% TFA. Elute peptidene med 200 mikroliter 0,1% TFA og 80% acetonitril.Etter å ha tegnet eluenten som demonstrert, resuspender den tørkede til fosfoproteomikkprøven i 200 mikroliter lastbuffer for fosfoptidanrikning. Tilsett 50 mikroliter av perlene i prøven og rist kraftig i 20 minutter ved romtemperatur. Legg prøven med perlene i den stekte spissen og sentrifugen i ett minutt ved 100 G.Vask spissen suksessivt med 200 mikroliter lastbuffer, vask buffere en og to.Plasser spissen med perler i et nytt rør for å samle de unnslupne fosfopenptidene. Tilsett 50 mikroliter elueringsbuffer til spissen og sentrifuge i to minutter ved 20 G.Tørk deretter de unnsluppede fosfoptidene ved hjelp av en vakuumsentrifugekonsentrator.

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 ...