Global chromatin dynamics have been shown to be an important mediator of several disease states, such as cancer and aging. Our protocol provides a physiologically relevant model to study this process. Standard laboratory analysis of histone post-translational modifications or PTMS is usually limited to probing for a single PTM at a time using antibody-based assays.
Liquid chromatography mass spectrometry analysis of histone PTMS allows us to measure the abundance of hundreds of PTMS in a single experiment. Demonstrating the procedure will be Stephanie Stransky, Ronald Cutler, and Julie Kim, a postdoc, grad student, and research tech, respectively, from the lab. First, using standard growth media, grow the cells as a monolayer until they are 80%confluent.
Then wash the cells with HBSS. Add five milliliters of 0.05%trypsin-EDTA diluted in HBSS. Incubate the cells for five minutes at 37 degrees Celsius with 5%carbon dioxide.
Use a microscope to check cell detachment. After counting the cells, dilute the cell suspension with fresh growth media. Add 0.5 milliliters of growth media to equilibrate an ultra low attachment 24-well round bottom plate with multiple micro-wells in each.
Then centrifuge the plates at 3000 x g to remove air bubbles from the surface of the plate. Now transfer the previously made cell suspension to the 24-well plate and centrifuge for three minutes at 120 x g. Check the cells in the 24-well plate and then incubate the plates at 37 degrees Celsius with 5%carbon dioxide for 24 hours for spheroid formation.
Meanwhile, to equilibrate the bioreactor, add 25 milliliters of sterile water to the humidity chamber and nine milliliters of growth media to the cell chamber. Incubate the bioreactor in a rotating clinostat incubator for 24 hours at 37 degrees Celsius with 5%carbon dioxide. Detach the spheroids from the ultra low attachment 24-well plate by gently pipetting using one milliliter wide bore tips, and transfer them to a tissue culture dish.
To select sufficiently forms spheroids, check the quality of spheroids under a microscope. Good quality spheroids have uniform size, compactness, and roundness. Fill the equilibrated bioreactor with five milliliters of fresh growth media, and transfer the spheroids into it.
Then fill the bioreactor completely with fresh growth media and put the bioreactor in the clinostat incubator at 10 to 11 RPM. Every two to three days, exchange growth media by replacing 10 milliliters of old media with fresh media. As this spheroids increase in size and number, increase the rotation speed of the incubator.
After obtaining the spheroid pellet, add five volumes of cold 0.2 molar sulfuric acid to the pellet and pipette up and down to disrupt the pellet and release histones. Once the supernatant is obtained after centrifugation, add cold concentrated trichloroacetic acid, such that the final concentration becomes 25 to 30%volume by volume. And mix it by inverting the tube a few times and centrifuge as described in the manuscript.
After discarding the supernatant, using a glass pasture pipette, wash the pellet and the walls of the tube with approximately 500 microliters of cold acetone and 0.1%hydrochloric acid, and then centrifuge at 3, 400 x g for five minutes at four degrees Celsius. Then flip the tube to discard the supernatant. Using a glass pasture pipette, add 500 microliters of 100%cold acetone to wash the pellet and centrifuge as demonstrated previously.
After discarding the supernatant, completely remove acetone by pipetting carefully, and let the sample dry with an open lid for about 20 minutes. Resuspend the pellet in 20 microliters of 15 to 20%of acetonitrile in a 100 millimolar ammonium bicarbonate of pH 8. After vortexing, spin down the suspension at 1000 x g for 30 seconds.
For eight or more samples, transfer each re suspended sample to a 96-well plate. Then, in a hood, add two microliters of propionic anhydride and mix by pipetting five times. Quickly add 10 microliters of ammonium hydroxide and mix by pipetting five times.
Mix HLB resin on a magnetic stir plate. Then add 70 microliters of the HLB suspension to each well of a 96-well filter plate on a 96-well collection plate. After discarding the flow through, wash the resin with 100 microliters of 0.1%trifluoroacetic acid.
After resuspending each sample in 100 microliters of 0.1%trifluoroacetic acid, load each sample in each well and prevent splashing using a vacuum. After discarding the flow through, wash the samples with 100 microliters of 0.1%trifluoroacetic acid, and put the filter plate on a new collection plate. Next, add 60 microliters of 60%acetonitrile in 0.1%trifluoroacetic acid to each well and prevent splashing using vacuum.
Collect the flow through and dry in a speed vacuum. Load the dried collection plate into the HPLC and run the LC/MS/MS method as described in the manuscript. In this study, the relative abundance of common histone post-translational modifications was observed in C3A hepatocytes grown as three dimensional spheroids.
Post-translational modifications could also be observed on a single residue in peptides generated from histone proteins. Treatment of the spheroids with sodium butyrate increased the relative abundance of histone acetylation, whereas that with sodium succinate enhanced histone residue succinylation. The method also demonstrated the distribution pattern of histone acetylation after sodium butyrate treatment and that of histone succinylation after treatment with sodium succinate.
The result also showed commonatorial patterns of different histone modifications. Sodium butyrate treatments significantly enhanced the acetylation of the lysine residue 14 on a peptide generated from the histone protein H3, only when its ninth lysine residue had two methyl groups, but not one or three methyl groups. The relative abundances of individual modifications and various combinations of post-translational modifications in the H3-derived peptide were also calculated.
Combinatorial patterns of post-translational modifications after sodium butyrate treatment were also observed in a peptide generated from histone protein H4.Here also, sodium butyrate treatment resulted in a significant increase in acetylation. Critical attention should be placed on keeping the spheroids on the bench as little as possible and in a desalting step to avoid sample spilling. This workflow can be used to explore chromatin in solid tissues using a more physiological model than fast replicating flat cell cultures.
