proteome/phosphoproteome analysis of the extracellular vesicle (ev) samples using lc-ms/ms

Enhancing EV Isolation for Precise Biofluid Analysis

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.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

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

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 Proteome/Phosphoproteome Analysis of the Extracellular Vesicle EV Samples Using LC-MS/MS at JoVE.com

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

To begin, prepare proteome and phosphoproteome samples of the extracellular vesicles isolated from the urine samples using the EVtrap approach. Inject the samples into the trapped ion-mobility time-of-flight mass spectrometer through the liquid chromatography system. Separate the peptides into a 15-centimeter C18 column.For proteomics analysis, acquire data using the dia-PASEF method with a mass range per ramp to span from 300 to 1200 mass-to-charge ratio and ion-mobility constant from 0.6 to 1.50 1/Knot. For phosphoproteomics analysis, acquire data using a dia-PASEF acquisition method. Set the mass range per ramp to span from 400 to 1550 mass-to-charge ratio in ion-mobility constant from 0.6 to 1.50 1/Knot.Load the raw files into proteomic software. Set up the search parameters in the homo sapiens database. For Digest Type, select Specific, and under Enzymes, select TrypsinP.Define the Peptide Length to a minimum of 7 and a maximum of 52 and allow two missed cleavages. Then set the Maximum Variable Modifications to 5. The Fixed Modification should be Carbamidomethyl at Cystine, while the Variable Modifications should include Acetyl Protein N-Terminal, Oxidation at Methionine, and Phosphorylation at Serine, Threonine, and Tyrosine.Finally, set the false discovery rate for peptide spectrum match, peptide, and protein group to 0.01. In the LCMS and MS proteomic profiling, 2%of each sample revealed over 11, 000 unique peptides from approximately 2, 200 proteins. Notably, 72%of unique proteins were consistently found in all three replicates and 90 top EV markers and proteins were identified compared to the ExoCarta database.Quantitative precision was confirmed with a low-medium coefficient of variation of 5.7%signifying high reproducibility and reliability. For phosphoproteomics, 98%of each peptide sample yielded 800 unique phosphopeptides and 350 phosphoproteins. The enrichment resulted in 72%phosphoserine, 22%phosphothreonine, and 6%phosphotyrosine peptides.42%of phosphopeptides were identified in all replicates in a medium coefficient of variation of 21.8%was observed, indicating acceptable quantitative reproducibility.

proteomephosphoproteome-analysis-extracellular-vesicle-ev-samples

Research

JoVE Journal

Biochemistry

241 Görüntüleme.  Purdue University. Başlamak için, EVtrap yaklaşımını kullanarak idrar örneklerinden izole edilen hücre dışı veziküllerin proteom ve fosfoproteom örneklerini hazırlayın. Numuneleri, sıvı kromatografi sistemi aracılığıyla sıkışmış iyon hareketliliği uçuş süresi kütle spektrometresine enjekte edin. Peptitleri 15 santimetrelik bir C18 kolonuna ayırın.Proteomik analiz için, rampa başına kütle aralığı 300 ila 1200 kütle-yük oranı ve 0,6 ila 1,50 1/Knot arasında iyon ...