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

We are working on accelerating based biomarker discovery. One important step is to isolate them efficiently and selectively. EV trap, which is based on Mag-Net EV-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 discovering proteins and the phosphoprotein biomarkers for different diseases such as spread cancer, kidney cancers, and the Parkinson's disease. This biomarkers derive 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 visco isolation are ultracintification and precipitation. But, this methods are limited to co-isolation of contaminants and also low efficiency. So, our EV trend 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 continuing developing methods and protocols for EV isolation and a downstream analysis, in particular, mass spec-based analysis. Second one is we are exploring different application in clinical field because of the advantage by the EV shot.

To begin, collect the urine samples from healthy individuals. Centrifuge 12 milliliters of sample in a 15 milliliter conical tube to eliminate cell debris in large apoptotic bodies. Then, transfer 10 milliliters of the supernatant into a fresh 15 milliliter tube.

Add one milliliter of loading buffer, and 200 microliters of EV trap bead slurry to the sample. Incubate the sample at room temperature for 30 minutes with end over end rotation. To pellet the sample, place the 15 milliliter conical tube on a magnetic separator rack.

Carefully remove the supernatant and resuspend the beads in one milliliter of washing buffer. Then, transfer the suspension to a 1.5 milliliter micro centrifuge tube and gently pipette to resuspend the extracellular vesicle or EV bound beads. Place the micro centrifuge tube on a magnetic separator rack for 1.5 milliliter tubes.

Using a P200 pipette, carefully aspirate the supernatant. Now, wash the beads with one milliliter of washing buffer, followed by two washes with one milliliter of PBS at room temperature. Incubate the beads with 100 microliters of freshly prepared 100 millimolar triethylamine for five minutes.

Using 1.5 milliliter micro centrifuge tube magnetic separator rack, collect the eluded solution containing EVs. After combining the eluded solutions, dry the Eluate using a vacuum centrifuge concentrator at four degrees Celsius. To begin, take the dried to extracellular vesicle sample obtained from the human urine using the EV trap approach.

Prepare a fresh lysis buffer with the shown components. Then, add 100 microliters of lysis buffer to solubilize the dried EV sample. Heat the sample at 95 degrees Celsius for 10 minutes while shaking at 1, 100 RPM.

After cooling the sample to room temperature, dilute it fivefold by adding 400 microliters of 50 millimolar TEAB. Measure the protein concentration using a BCA assay kit according to the manufacturer's instructions. Add trypsin and lysine C mix to the sample.

And incubate at 37 degrees Celsius overnight with shaking at 1, 100 RPM. Then, add 50 microliters of 10%trichloroacetic acid or TFA to acidify the sample. Further, add 600 microliters of ethyl acetate to the samples and vortex the mixture for two minutes.

Centrifuge the sample for three minutes at 20, 000 G and carefully remove the upper layer without disturbing the interface. Dry the aqueous phase using a vacuum centrifuge concentrator. Now, resuspend the dried sample in 200 microliters of 0.1%TFA to acidify peptides.

To desalt the sample, use a C18 desalting tip and condition it with 200 microliters of 0.1%TFA and 80%acetonitrile, followed by two washes with 200 microliters of 0.1%TFA. Load the acidified peptide sample into the tip and wash it three times with 200 microliters of 0.1%TFA. Elute the peptides with 200 microliters of 0.1%TFA and 80%acetonitrile.

After drawing the eluate as demonstrated, resuspend the dried phosphoproteomic sample in 200 microliters of loading buffer for phosphopeptide enrichment. Add 50 microliters of the beads to the sample and vigorously shake for 20 minutes at room temperature. Load the sample with the beads into the fritted tip, and centrifuge for one minute at 100 G.Wash the tip successively with 200 microliters of loading buffer, washing buffers one and two.

Place the tip with beads into a new tube to collect the eluded phosphopeptides. Add 50 microliters of elution buffer to the tip. And centrifuge for two minutes at 20G.

Then, dry the eluded phosphopeptides using a vacuum centrifuge concentrator. To begin, prepare proteome and phosphoproteome samples of the extracellular vesicles isolated from the urine samples using the EV trap 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 1.501 over K 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 and ion mobility constant from 0.6 to 1.501 over knot. Load the raw files into proteomic software. Set up the search parameters in the homosapiens database.

For digest type, select specific and under enzymes, select trypsin P.Define the peptide length to a minimum of seven and a maximum of 52 and allow two missed cleavages. Then, set the maximum variable modifications to five. The fixed modification should be carbamidomethyl at cysteine.

While the variable modifications should include:acetyl protein and 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%phosphoseine, 22%phosphothreonine, and 6%phosphotyrosine peptides. 42%of phosphopeptides were identified in all replicates and a medium coefficient of variation of 21.8%was observed indicating acceptable quantitative reproducibility.