The overall goal of this experimental strategy is to resolve the distinct functional assemblies a protein may be involved in through blue native polyacrylamide gel electrophoresis fractionation of affinity purified protein complexes and mass spectrometry correlation profiling. This method can help add organizational information to the protein lists generated by affinity purification massive spectrometry experiments. The main advantages of this procedure are that is simple to perform, has good resolution, and it takes little more work than conventional gel CMSMS.
This protocol is optimized for the purification of protein complexes from 200 to 500 million cells expressing endogenous levels of a FLAG tagged protein. After thawing the cell pellet, as described in the text protocol, add five milliliters of ice cold lysis buffer containing DTT and protease inhibitors to the cell suspension and swirl to mix. Incubate the suspension on ice for 10 minutes.
Transfer the cell suspension to a cold down homogenizer. Lyse the cells with 20 to 30 strokes, using the tight pestle until there is no noticeable viscosity, then transfer the homogenate to cold micro centrifuge tubes. After centrifuging the tubes, transfer the cleared lysate to a clean cold tube, leaving approximately 50 microliters of lysate behind.
Save a 35 microliter aliquot of lysate to measure protein concentration and monitor the purification. Next, remove the buffer from the previously prepared beads. Resuspend the beads with one milliliter of the lysate, and then mix with the rest of the lysate.
Incubate the mixture on a rotating wheel at four degrees Celsius for one to two hours. Collect the beads on the side of the tube with a magnet. Collect a 30 microliter aliquot of supernatant and discard the rest.
Resuspend the beads in 0.5 milliliters of IPP150 buffer by pipetting four to six times. Then, transfer the suspension to a 1.5 milliliter cold tube. Next, wash the beads three times with 0.5 milliliters of FLAG elution native buffer.
With the last wash, transfer the beads to a new cold tube. Remove the buffer thoroughly. Resuspend the beads in 100 microliters of 200 micrograms per milliliter 3X5 peptide in native elution buffer.
Then, incubate at four degrees Celsius for 10 minutes with gentle rotation. Collect the beads with the magnet, and transfer the supernatant to a cold new tube. Repeat this step twice before pulling all the eluates.
Concentrate the eluate down to 25 microliters in a centrifugal filter unit by centrifugation at 10, 000 times G at four degrees Celsius. Prepare the sample by adding 4X native sample loading buffer and 0.5%G-250 sample additive to the 25 microliter concentrated eluate. Load the sample and native molecular weight markers, leaving an empty well between them.
Then, load 10 to 20 microliters of 1X native sample loading buffer in all the empty wells. Fill the cathode chamber with 200 milliliters of 1X native PAGE dark blue cathode buffer carefully, so as not to disturb the samples. Fill the anode chamber with 550 milliliters of 1X native PAGE anode buffer.
Run the gel at 150 volts for 30 minutes. After stopping the run, remove the dark blue cathode buffer with a serological pipette and replace with 1X light blue cathode buffer. After continuing the run for 60 minutes, open the gel cassette and discard one of the cassette plates.
The gel remains attached to the other cassette plate. Place the gel in a tray containing enough fixative solution to cover it, and incubate for 30 minutes with gentle shaking. Then, remove the fixative solution and replace with water before scanning the gel.
To prepare for mass spectrometry, first place a conical bottom 96 well plate on top of another 96 well plate to collect the waste. Pierce the bottom of the wells of the conical bottom 96 well plate with a 21 gauge needle. Then, add 200 microliters of 50%acetonitrile, 0.25%formic acid to the wells of the pierced plate.
Incubate the plate stack in a shaker for 10 minutes. Next, centrifuge the plate at 500 times G for one minute, so that the liquid flows through the holes into the waste collection plate. After repeating the washing steps twice, fill the wells of the pierced 96 well plate with 150 microliters of 50 millimolar ammonium bicarbonate.
Next, place the gel on a clean surface. Slice the lane containing the sample into 48 identical slices. Cut each slice into two or three smaller pieces, except the last five slices at the top of the gel.
Place each slice sequentially in one well of the pierced end washed 96 well plate. Add 50 microliters of acetonitrile to each well and incubate the plate with shaking for 30 to 60 minutes. After removing the liquid by centrifugation, add 200 microliters of two millimeter TSEB and 50 millimolar ammonium bicarbonate to each well.
Incubate the plate with shaking for 30 minutes at room temperature before removing the liquid by centrifugation as before. Dehydrate the gel pieces by adding 200 microliters of pure acetonitrile and incubate with shaking at room temperature for 20 minutes until the gel pieces are white and opaque. After removing the acetonitrile, add 150 microliters of trypsin in cold ammonium bicarbonate to each well.
Incubate the plate with shaking at 37 degrees Celsius for two hours. Next, place a clean conical bottom plate under the gel containing plate, ensuring correct orientation of the plates. Then, collect the liquid containing the peptides by centrifugation at 200 times G for one minute.
Place the peptides containing plate in a centrifugal evaporator and evaporate the liquid whilst performing the next step. Add 150 microliters of 50%acetonitrile, 0.25%formic acid to the gel pieces. Incubate with shaking at 37 degrees Celsius for 30 minutes.
Collect the liquid into the partially dried plate by centrifugation at 200 times G for one minute. Continue evaporating the peptides containing plate whilst performing the next step. Transfer the peptide solutions to the washed centrifugal filtration plate.
Place a clean conical bottom plate underneath it to collect the peptides and centrifuge the plate stack as before. Evaporate the liquid in the conical bottom plate until the wells are completely dry. Once dry, the plate can be covered with a silicone lid and stored at minus 20 degrees Celsius until performing mass spectrometry and data analysis as described in the text protocol.
Representative results from the hierarchical clustering of blue native PAGE migration profiles of Mta2 and copurifying proteins are shown here as a dendrogram and a heat map representing protein intensities. The migration profile of Mta2 shows two peaks indicative of two distinct complexes. Some NuRD subunits show a similar pattern with highest abundance in the lower molecular weight assembly.
By contrast, other NuRD subunits are more abundant in the higher molecular weight NuRD assembly. On the other hand, the Mta2 interacter, Cdk2ap1, is only present in the higher molecular weight NuRD assembly. Once mastered, this technique can be done in two and a half days.
While attempting this procedure, it's important to remember to keep all samples and reagents on ice to avoid dissociation of protein complexes. This procedure can be applied to any protein of interest, provided it can be looked at from the affinity medium by competitive elution in native conditions. Following this procedure, other methods like reverse or sequential co-immunoprecipitation can be performed to validate the identified subcomplexes.
