Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

The overall goal of this protocol is to provide a simple procedure to assess the relative abundance of histone post-translational modifications, which play essential roles in chromatin biology, such as gene expression regulation and chromosome condensation. This method can help answer key questions in the chromatin biology field. Such as, which histone modifications change their relative abundance on stimulation or treatment of given cells or tissue.

The main advantage of this technique is that, by using mass spectrometry-based proteomics, we can simultaneously quantify most of the known PTMs on known proteins in a single analysis. Histone modification analysis has numerous applications, including estimating epigenetic changes and chromatin operation of individuals or model system upon drug treatment or stress. In addition to Simone, two other members of my laboratory will demonstrate the procedures of this protocol.

Natarajan Bhanu, a research specialist, and Kelly Karch, a graduate student. As shown in this workflow, this is a 10-step protocol that includes histone purification from cell cultures or tissues, histone derivatization, digestion and desalting, and mass spectrometry analysis using nanoflow liquid chromatography. Only selected procedures will be demonstrated in this video.

To begin the procedure for isolating nuclei from intact cells, thaw on ice, cells that were previously harvested and stored at negative 80 degrees Celsius. And prepare the nuclear isolation buffer, or NIB, as described in the protocol text. Remove one fifth volume of NIB and add NP-40 Alternative to a final concentration of 0.2%The remaining four-fifth volume will be used for washes.

Wash the cell pellet with NIB without NP-40 Alternative, using a 1:10 ratio of cell pellet volume to buffer volume. Centrifuge at 700 RCF for five minutes and remove the supernatant. Lyse the cell pellet by placing in on ice and adding NIB with 0.2%NP-40 Alternative at a 1:10 ratio of cell pellet volume to buffer volume.

Homogenize the cells by gentle pipetting. Incubate the homogenized cells on ice for five to 10 minutes for the cells to lyse and release the nuclei. Centrifuge at 1000 RCF for five to 10 minutes at four degrees Celsius.

The pellet will contain mostly cell nuclei, while the supernatent will contain mostly cytoplasmic components. Save the cytoplasmic fraction if desired. Wash the nuclei pellet by gently re-suspending it NIB without NP-40 Alternative, using a 1:10 ratio of pellet volume to buffer volume.

Centrifuge at 1000 RCF for five minutes at four degrees Celsius, and remove the supernatent. After NP-40 Alternative has been completely removed from the nuclei pellet, add ice-cold 0.2 molar sulfuric acid at a 1:5 ratio of nuclei volume to sulfuric acid volume. Re-suspend the nuclei in the sulfuric acid by gentle pipetting.

Incubate the sample with constant rotation, or gentle shaking for two to four hours at four degrees Celsius. Next, centrifuge the sample at 3400 RCF at four degrees Celsius for five minutes and transfer the supernatent to a new tube. Centrifuge again and transfer the supernatent to a new tube to remove any insoluble material.

To precipitate the histones, add chilled, 100%trichloroacetic acid, or TCA, to the collected supernatent in a 1:3 volume ratio. The presence of histones is indicated by the sample turning cloudy. Mix by inverting the tube a few times.

Incubate the mixture on ice for at least one hour. Centrifuge at 3400 RCF for five minutes. After centrifugation, the histones will coat the sides of the tube and also deposit at the bottom.

A white, insoluble pellet will also form at the very bottom of the tube, which mostly contains non-histone proteins and other bio-molecules. Carefully remove the supernatent by aspiration, without scraping the sides of the tube, or the pellet at the bottom of the tube. By using a glass Pasteur pipette, rinse the tube with 100%ice-cold acetone plus 0.1%hydrochloric acid, so as to cover the precipitated proteins coating the sides and bottom.

Centrifuge at 3400 RCF for two minutes. Aspirate the supernatent carefully, without scraping the sides or the pellet. Rinse the tube with 100%ice-cold acetone, centrifuge again, and remove the supernatent without scraping the sides or the pellet.

Subsequently, histone quantification and purity analysis is performed as described in the protocol text. An optional fractionation of histone variants by reversed-phase HPLC can also be performed before histone derivatization. Begin this procedure by dissolving the histone samples in 40 microliters of 50 millimolar ammonium bicarbonate, pH 8.0.

Check the pH by inserting a P10 pipette tip into the sample without aspiration, and touching the pipette tip to a pH indicator strip. Use ammonium hydroxide and formic acid to adjust the pH to 8.0. From here on, all steps involving the use of propionic anhydride must be performed in a fume hood.

Process a maximum of three to four samples at a time, in order to keep propionic anhydride reactive. Prepare fresh propionylation reagent by mixing propionic anhydride with acetonitrile in a volume ration of 1:3. Add the propionylation reagent to each sample in a volume ratio of 1:4.

Quickly add ammonium hydroxide to re-establish pH 8.0 to the solution. Usually, adding ammonium hydroxide to the sample with a volume ratio of 1:5 is appropriate to re-establish pH 8.0. Mix immediately by vortexing.

Check the pH. Incubate the samples at room temperature for 15 minutes. After all samples have undergone propionylation, dry the samples down to 10 to 20 microliters in a vacuum concentrator.

Re-suspend or dilute samples with ddH20 until 40 microliters of final volume is achieved. Since salts impede liquid chromatography and mass spectrometry, the samples must be desalted prior to the analysis. For the purposes of this video, only the construction of the stage-tips will be demonstrated.

Use a P1000 pipette tip to punch a disk of C18 material from a solid phase extraction disk. Use a fused silica capillary to push the mini-disk out of the P1000 tip and into the bottom of a P100 or P200 pipette tip. Ensure that the disk is securely wedged at the bottom of the tip.

If desalting over 25 micrograms of sample, use two C18 mini-disks in the same P100 or P200 tip. Use a centrifuge adaptor to hold the stage-tips in place in 1.5 or two milliliter microcentrifuge tubes. Flush the resin by spinning with 50 microliters of 100%acetonitrile to activate the C18 material and remove potential contaminants.

Sample desalting is subsequently performed as described in the protocol text. Histone peptides are present in a variety of isobaric forms. Two examples commonly abundant in histone analysis are shown.

The extracted ion chromatogram of their precursor mass and relative isotopes, shown above, is identical. However, the extracted ion chromatogram of the product ions, shown below, allows for discrimination of the two isobaric forms. Only unique fragment ions, highlighted in red, should be used to estimate the relative abundance of the two species.

Histones extracted from human embryonic stem cells, with and without retinoic acid stimulation, were analyzed. Relative quantification of two histone H3 peptides revealed a clear reduction of acetylation in human embryonic stem cells, stimulated for differentiation. This is consistent with previous reports of higher acetylation in embryonic stem cells, compared to differentiating cells.

Once mastered, this technique can be done in three days if performed properly. One day for histone extraction, the second day for protein derivatization and digestion and the third day for peptide derivatization, stage-tipping and LC-MS analysis. Histone purification can be exploited for other purposes other than analysis of peptides, including middle-down and top-down proteomics, where it's possible to charactarize combinatorial histone modifications.

After watching this video you should have a good understanding of how to identify and quantify histone post-translational modification using mass spectrometry based proteomics.