This method can help answer key questions in any research field where phosphorylation signal is of interest such as the cancer field to asses changes in signaling networks after therapeutic resistance. The main advantage of this technique is that thousands of phosphopeptides can be identified in a relatively simple and inexpensive way to globally assess the phosphoproteome. To harvest tumor tissue, weigh the tumor and in a culture test tube, add two milliliters of ice cold lysis buffer for every 100 milligrams of tissue.
Then use a handheld or bench top homogenizer to homogenize the lysate. To reduce and alcoholate the sample, heat the tube at 95 degrees Celsius for five minutes, then cool the sample on ice for 15 minutes. While still on ice, sonicate the lysate three times, then heat the sample at 95 degrees Celsius for five minutes.
Place the sonication tube in a swinging bucket rotor and centrifuge the lysate at 3, 500 g and 15 degrees Celsius for 15 minutes, then collect the supernatant and discard the pellet. To digest the lysate, use 100 millimolar tris to dilute the sample 12-fold to reduce the amount of guanidinium. For five milligrams of protein, add 10 micrograms of lysyl endopeptidase or lys-C and incubate the sample at room temperature for five to six hours.
Prepare one milligram per milliliter of TPCK-treated trypsin in one millimolar HCL with 20 millimolar calcium chloride and trypsin added at a one to 100 trypsin to protein ratio. Incubate the sample at 37 degrees Celsius for three hours. Then add an additional aliquot of trypsin to the tube and incubate the sample at 37 degrees Celsius overnight.
Add the sample to a 15 milliliter 10 kilodalton cutoff filter. Centrifuge the sample in a swinging bucket or fixed angle rotor at 3, 500 g and 15 degrees Celsius until the retentate volume is less than 250 microliters. Then collect the flow through and discard the retentate.
To acidify the sample, add approximately 20 microliters of 5%Trifluoroacetic Acid or TFA per milliliter of lysate. Mix the tube well and use a pH strip to measure the pH. If necessary, add extra 5%TFA to adjust the pH to 2.5.
Next, connect to the shorter end of a C18 column to a vacuum manifold. After the setting the vacuum between 17 and 34 kilopascals, use three milliliters of 100%acetonitrile and a glass pipette to wet the column. Equilibrate the column by using a glass pipette and applying two three milliliter aliquots of 0.1%TFA.
Then load the acidified sample onto the column. Use three three milliliter volumes of 0.1 TFA to wash the column. Then apply two milliliters of 40%ACN 0.1%TFA to elute the sample and collect two two milliliter fractions into glass culture tubes.
With parafilm, cover the eluent tubes and use a 20 gauge needle to punch three to five holes into the cover before lyophilizing the samples overnight. Resuspend the lyophilized powder with 0.5 milliliters of ice cold immunoprecipitation or IP binding buffer in each fraction. Transfer the 0.5 milliliter resuspension volume from the second fraction to the tube with the first fraction and save the pipette tip.
Use another 0.5 milliliters of IP buffer to rinse each lyophilization tube before transferring the contents to the cryotube, then repeat the rinse using two milliliters of IP buffer. After using a P200 with a cut tip to transfer the 0.5 milligrams per milliliter stock of antibody bead slurry to the microfuge tube, add 450 microliters of ice cold IP binding buffer to wash 50 microliters of 4G10 antibody bead slurry and 25 microliters of 27 B10.4 antibody bead slurry in a microfuge tube. Centrifuge the tube at 100 g and four degrees Celsius for one minute.
After aspirating the supernatant, add an equal volume as the original slurry of IP binding buffer to resuspend the beads. Add prewashed pY beads to the resuspended sample solution in the screw cap cryotubes, then incubate them at four degrees Celsius on an end-over-end rotator overnight. After spinning down the beads, save the supernatant which will be used to enrich for pST peptides.
Next, use 300 microliters of IP binding buffer to resuspend the beads and transfer them to a two milliliter microcentrifuge tube. Spin down the samples at 100 g and four degrees Celsius for one minute. Then with 500 microliters of IP binding buffer, wash the beads three times.
Wash the beads four times with 450 microliters of 25 millimolar ammonium bicarbonate pH 7.5. Dip a gel loading tip slightly below the bead surface and completely aspirate the supernatant. Add four times the bead volume of 0.1%TFA to the beads.
Then mix them well and incubate the tube in a thermomixer at 1, 000 rpm and 37 degrees Celsius for 15 minutes. Transfer the resuspension to a 0.2 micrometer spin filter and centrifuge the filter at 850 g for one minute. After transferring the elution to a low-protein binding microcentrifuge tube, vacuum concentrate the eluent to dryness at 40 degrees Celsius with a heat time of 300 minutes overnight.
To prepare the titanium oxide beads contained in tips, add 200 microliters of 100%ACN. Then invert it and flick the narrow end to move the liquid towards the cap. Using a razor blade, cut the tip and place it over the low-protein binding tube.
Then remove the cap and insert a micropipette to plunge out the remaining ACN before repeating the wash. Precondition the titanium dioxide with 500 microliters of 100%ACN two times. Then condition the titanium dioxide with 500 microliters of 0.2 molar sodium phosphate buffer pH 7 two times.
Finally, use 300 microliters of equilibration buffer to wash the beads three times. Add 400 microliters of 50%ACN 0.1%TFA to the low-protein binding tube. Then add 84 microliters of lactic acid.
Transfer the resuspended phosphopeptides into the low-protein binding tube and incubate them at room temperature using an end-over-end rotator for one hour. After pelleting the beads, use 300 microliters of equilibration buffer to wash them two times and spin them down. With 300 microliters of rinsing buffer, rinse the beads two times.
Then transfer them to a 0.2 micrometer spin filter. After spinning, transfer the filter unit to a clean 1.5 milliliter low-protein binding tube and with 200 microliters of 0.9%ammonium in water, elute the contents two times. After checking the pH, vacuum concentrate the eluent to dryness overnight to evaporate the ammonia.
To desalt the peptides for MS analysis, reconstitute the phosphopeptides with 15 microliters of 0.1%TFA. Clean the sample using a C18 tip with a binding capacity of five micrograms and follow the manufacturer's protocol. Finally, after completely drying the elution volume by vacuum concentration, resuspend the dried phosphopeptides in 12.5 microliters of mass spectrometry solution.
This Coomassie-stained gel of predigested lysate confirms the presence of proteins while staining of the post-digested lysate confirms the complete digestion. For complete digestion, no bands should appear above 15 kilodaltons except the 30 kilodalton and 23.3 kilodalton bands for lys-C and trypsin respectively. The addition of lys-C also reduces the number of missed cleavages.
The pY immunoprecipitation effectively separates pY from pST peptides were on average 85%of the phosphopeptides identified from the pY preparation are pY and over 99%of the phosphopeptides identified from the pST preparation are pST. Titanium dioxide is used to enrich for phosphopeptides in both preparations. The expected percentage of peptides in the MS ready preparation that are phosphorylated is between 30 to 50%and the majority of the phosphopeptides detected have a single or double phosphoryl group.
After performing mass spectrometry, the MS raw files are loaded into an MS analysis software. As illustrated here, setting a localization probability cutoff of greater than 0.75 filters out approximately 5%of the pY peptides and 15%and 34%of the pS and pT peptides respectively. After applying these filters, the expected number of phosphopeptide identifications at the end of the MS analysis is approximately 300 pY peptides for five milligrams of the starting protein and about 7, 500 pS peptides and 640 pT peptides for 2.5 milligrams of the starting peptide amount from the respective enrichment preparations.
Once mastered, this technique can be done in six days if the pY and pST steps are performed in parallel. However, for more than eight samples, perform the steps in sequence to reduce technical error over an extra four days. While attempting these procedures, it's important to remember to focus on the details.
Every step is important and can affect the quality of the final preparations including the necessary quality controls is critical. After watching this video, you should have a good understanding of how to extract and digest proteins, followed by phosphopeptide enrichment for analysis via mass spectrometry to investigate global changes in the phosphoproteome.
