The overall goal of this procedure is to purify vesicles from blood while reducing co-purifying proteins and complexes which can interfere with downstream analysis including nanoparticle tracking and proteomics. This method can help answer key questions such as what are the proteomic changes in serum or plasma vesicles during the disease state as compared to a healthy state. The main advantage of this technique is the increased accuracy in vesicle enumeration.
In removing abundant non-vesicle contaminants, we increase the identification of low abundance proteins and the potential biomarkers of disease. The implication of this method extend to the discovery of novel diagnostic biomarkers. It enables more accurate determination of the proteomic composition of extracellular vesicles in complex matrices, like blood.
Generally, individuals new to vesicle purification will struggle because there are many published techniques. The reproducability of downstream proteomic analysis is affected by the co-purification of abundant serum proteins. Equilibrate a serum or plasma sample by placing the tube under non-shaking conditions in a four degree Celsius refrigerator overnight.
The next day, aliquot 250 microliters of the sample into a microcentrifuge tube using a 1, 000 microliter pipette. Centrifuge the sample at 18, 000 times G for 30 minutes at four degrees Celsius. Using a 200 microliter pipette, remove 200 microliters of the cleared supernatant and add it to a new microcentrifuge tube.
Avoid any pelleted material while transferring the supernatant. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol. To perform the assay, dilute the sample one to 50 in one X phosphate buffered saline.
Test each sample in duplicate. Prepare a proteinase K solution at a concentration of 50 micrograms per milliliter in PBS by first adding the corresponding amount of proteinase K to a conical tube. Then add the buffer and vortex for 30 seconds three times at room temperature.
Next, add 50 microliters of proteinase K solution to the 10 milligram sample aliquot and mix gently by pipetting up and down using a 200 microliter pipette. Incubate the sample at 37 degrees Celsius in a water bath for 30 minutes, placing the tube in a float tube rack. To prevent potential leakage, avoid complete immersion of the tube in the water bath.
Following incubation, transfer the sample and rack to a 60 degree Celsius water bath for 10 minutes to inactivate the proteinase K.Next, rinse an ultrafiltration device containing a 100 kilodalton molecular weight cutoff filter with PBS prior to use. Use a 1, 000 microliter pipette to add 500 microliters of PBS to the filter. After spinning the device at 3, 700 times G for five minutes, discard any remaining retentate as well as the eluate.
Increase the volume of the sample to 500 microliters by adding PBS. Then pipette the sample into the pre-rinsed ultrafiltration device. Centrifuge the ultrafiltration device in a fixed angle benchtop centrifuge at 3, 700 times G and four degrees Celsius until the sample has reduced to a volume of 50 microliters.
Next, add 500 microliters of PBS directly onto the filter membrane. And pipette up and down five times before centrifuging again as before. Transfer the final 50 microliter retentate to a new microcentrifuge tube.
Rinse the filter membrane with 200 microliters of PBS, pipetting up and down 10 times. And transfer the wash to the tube. Then, place the filter holder in the inverse position in a new tube.
Run a reverse spin recovery for five minutes at 2, 000 times G to retrieve the remaining sample from the membrane. Place the columns in a size-appropriate rack. Add 850 microliters of SEC slurry with beads having a molecular weight cutoff of 700 kilodaltons using a 1, 000 microliter pipette.
Uncap the bottom of the column and let the liquid drain into a waste container for five minutes. Once drained, add 10 microliters of PBS to wash the resin. Allow 20 minutes for the wash to drain from the column.
Place the concentrated proteinase K treated sample into a 15 milliliter conical tube. And increase the sample volume to five milliliters by adding PBS with a serological pipette. Mix the tube gently by rocking for one minute.
Then, slowly apply the sample to the prepared column with a serological pipette. Collect the flow-through in a new 15 milliliter conical tube. Pipette the collected material over the resin again to remove any remaining material below 700 kilodaltons, collecting the flow-through in a new 15 milliliter conical tube.
Wash the resin twice by adding one milliliter of PBS each time. Collect the flow-through in a tube. The total final volume will be roughly seven milliliters.
Rinse an ultrafiltration device with a three kilodalton molecular weight filter as before. Centrifuge in a swinging bucket rotor at 3, 700 times G and four degrees Celsius. Buffer will pass through the filter and can be discarded.
Concentrated exosomes will be retained above the filter. Centrifuge the sample until the volume above the filter is reduced to 200 microliters. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol.
Add five micrograms of purified exosomes to one milliliter of PBS. And vortex for 15 seconds on low medium speed. Place the sample in a one milliliter disposable syringe.
If available, set the syringe in an automatic syringe pump set to inject the diluted exosome sample at a rate of 30 microliters per minute. Define the script for analysis to run three technical replicates for a minimum of 30 seconds using a constant flow for every replicate. For the analysis, set the capture threshold at five.
Finally, determine the soluble protein reduction as described in the text protocol. Representative results of nanoparticle tracking analysis of purified serum vesicles are shown. The vesicle size is roughly 100 nanometers, as expected.
Shown here is a Coomassie-stained protein gel comparing the crude serum, pre and post SEC fractions showing the reduction in albumen and other non-vesicle contaminants. A Western blot displays the retention of CD63, a hallmark exosome protein after proteinase K treatment and purification. The proteinase K step is designed to digest soluble proteins.
CD63 is a transmembrane protein and less susceptible to proteinase K at the determined concentration and incubation length. Here, a Western blot shows the depletion of albumen, the most abundant protein in blood and a common co-purifying protein after purification. Once mastered, this technique can be done in six hours.
After watching this video, you should have a good understanding of how to use centrifuge ultrafiltration, protease digestion, and size exclusion chromatography to obtain vesicles from biological fluids. It's important to perform protein quantitation by BCA or equivalent technique prior to a chromatography and nanoparticle tracking. Results will vary if protein concentrations exceed those noted in this protocol.
Don't forget that working with biological fluids can be extremely hazardous and precautions, such as wearing personal protective equipment including gloves, lab coat, and safety glasses should always be taken while performing this procedure. Following this procedure, other methods like liquid chromatography mass spec can be performed in order to determine the exact proteins contained in the vesicles purified. Though this method can provide insight into the composition of blood vesicles, it can also be applied to other sample types, such as urine, CSF, or cell culture supernatants.
