These protocols are designed to maximize the recovery as well as the purity of proteins through solvent-based precipitation. We're gonna show you a couple tricks to perform precipitation in vials, as well as a semi-automated approach in a spin cartridge format. We have also optimized the precipitation protocol to be consistent and quick.
The whole protocol only takes a few minutes. These protocols demonstrate that solvent precipitation of proteins is ideal for sample prep ahead of mass spectrometry. The protocols will fit seamlessly into bottom up as well as top down pro workflows.
Demonstrating for us today will be Ziheng Dang and Victoria Miller, Senior scientist at Proteoform Scientific in Halifax, Nova Scotia Pipette 90 microliters of particulate free protein solution into a polypropylene micro centrifuge tube. Then add 10 microliters of one mole aqueous sodium chloride and 400 microliters of acetone into the sample solution, cap and tap the vial gently to combine the solvents. Allow the vial to incubate it.
Room temperature undisturbed for a minimum of two minutes following incubation, centrifuge the samples noting the orientation of the vial and spin for a minimum of two minutes at 10, 000 RCF or higher at room temperature. After the centrifugation, a visible white pellet is observed at the bottom of the tube. At this point, uncap the tube and decant the supernate into a waste container by inverting the tube.
Use a paper towel to draw any residual solvent from the vial. For SDS containing samples. Carefully dispense 400 microliters of fresh acetone without disturbing the pellet.
Immediately centrafuse the sample for one minute at 10, 000 times G or higher at room temperature by placing the vial into the rotor in the same orientation as the initial spin. Decount the wash solvent as described previously. Dry the sample with the cap open for approximately one minute.
In a fume hood, before a pellet solubilization. Dispense 100 microliters of the protein solution into a polypropylene vial. In the fume hood, add a 400 microliters of methanol followed by 100 microliters of chloroform.
Cap the vial and vortex briefly to mix. Quickly dispense 300 microliters of water directly into the center of the vial. Cap the vial and allow it to sit on the bench top undisturbed for one minute, after place the polypropylene vial in a centrifuge and spin it for five minutes at 10, 000 times G or higher at room temperature.
The vial is observed to have two solvent layers and a visible white pellet til the vial at approximately 45 degrees. Remove approximately 700 microliters of the solvent from the upper layer at a uniform rate using a large one milliliter micro pipette tip initially and later with a 200 microliter pipette tip until it forms a bead in the vial. Add 400 microliters of fresh methanol to the sample vial without disturbing the pellet by dispensing the solvent down the side of the vial.
Cap the vial and combine the solvent layers by gently rocking the vial to swirl the solvents together. To observe the white protein pellet noting the orientation of the vial in the rotor centrifuge for a minimum of 10 minutes at 10, 000 times G or higher at room temperature. Angle the vial with the pellet facing down.
Place a pipette tip along the upper edge of the vial and remove the supernatant with a one milliliter micro pipette tip at a slow but uniform rate. Retaining approximately 20 microliters of solvent. Allow the residual solvent to dry in the fume hood.
To precipitate the protein. Using a disposable filtration cartridge attach the plug to the upper filtration cartridge. Combine 90 microliters of protein solution 10 microliters of one mole aquas, sodium chloride and 400 microliters of acetone.
Incubate for a minimum of two minutes on the bench top, centrifuge the prepared protein sample with the plug still attached to the filtration cartridge for two minutes at 2, 500 times G.At room temperature, invert the cartridge and unscrew and remove the plug from the cartridge base. Place the filtration cartridge in a clean vial centrifuge the protein sample for three minutes at 500 times G at room temperature. Discard the flow through solvent from the lower vial.
Wash the protein pellet by adding 400 microliters of acetone to the filtration cartridge, centrifuge for three minutes at 500 times G at room temperature or until no solvent remains in the upper cartridge. Following the previously demonstrated protein precipitation protocols. Re solubilize the protein by wetting the membrane at the base of filtration cartridge by dispensing two to five microliters of ISO propanol directly to the membrane immediately before proceeding to the re solubilization protocol for re solubilization of the protein pellet prepare 80%volume by volume solution of formic acid in water.
Pre chill this acid solution at minus 20 degrees Celsius and the filtration cartridge containing precipitated protein. Dispense 50 microliters of cold diluted formic acid into the chilled cartridge cap and vortex for 30 seconds. Return the filtration cartridge to the freezer at minus 20 degrees Celsius for 10 minutes.
Repeat the previous vortex and incubation steps. Add 450 microliters of water to a final volume of 500 microliters. Diluting the formic acid to 8%Invert the cartridge unscrew and remove the plug from the cartridge base and attach the SPE cartridge to the vial.
Spin the protein through the SPE cartridge in a centrifuge at room temperature for five minutes at 800 times G.If solvent remains in the upper cartridge return the cartridge to the centrifuge and repeat the spin. Rinse by adding 300 microliters of 5%acetonitrile with 0.1%of TFA in water to the SPE cartridge, flow through the SPE cartridge for two minutes at 2000 times G.For low molecular weight proteins or digested peptides elute the sample by flowing 300 microliters. A 50%acetonitrile with 0.1%of TFA for five minutes at 2, 500 times G.For intact proteins.
Follow up with an additional elusion step using 300 microliters of 75%acetonitrile with 0.1%Trifluoro ascetic acid. Combine the two resulting extracts. The figure compares the SDS depletion following vial based or precipitation of proteins in a disposable filter cartridge.
Using acetone with the conventional rapid and CMW protocols. All approaches reduce SDS to permit optimal mass spectrometry analysis. This figure shows quantitative protein recovery by following rapid acetone precipitation and CMW precipitation through SDS page analysis of a processed yeast total cell lysate.
Consistent recovery was obtained with all precipitation protocols. Precipitation in a disposable filtration cartridge eliminates the need to carefully pipette SDS containing supernatant while retaining the aggregated proteins above a membrane filter. Thus no visible bands were detected in the supernatant fractions across three independent replicates.
The cartridge based protein precipitation demonstrates superior recovery of a Pepsin digested sample of bovine plasma relative to vial based precipitation. More significant differences in yield are noted in the cartridge when employing sodium chloride while employing zinc solvent resulted in the highest yield. This figure quantifies the peptide peak intensity from each peptide sample with a ratio above one reflecting a higher peptide abundance for samples processed in the disposable filter cartridges.
The figure compares the number of identified peptides per protein across the three replicate sample preparations. The correlation coefficients of 0.94 to 0.95 in these graphs demonstrate the high consistency of the sample preparation approach for bottom up mass spectrometry analysis. So once that pellet is formed you really wanna avoid disturbing it.
If you keep it as an intact pellet you'll get a higher yield. So make sure not to forget to wash that pellet with extra acetone. This is making sure that we're getting rid of the SDS keeping our sample as clean as possible and that gives us the best mass spectrometry analysis.
So we found that removing the excess solvent by inverting the is a lot simpler than trying to pipette the sample. You'll get more consistent yields this way. Performing acetone precipitation at room temperature resulted in more consistent and faster recovery than traditional precipitation in the freezer.
