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

1. Collection of Cells from Culture

1. If cells were grown in suspension, collect cells by centrifugation at 300 rcf for 5 min. If adherent, aspirate and discard cell medium. Rinse the attached cells with PBS without Ca²⁺ and Mg²⁺ (referred to as PBS going forward). Incubate the cells in either trypsin or trypsin-EDTA (0.025% – 0.5% depending on the cell line) with enough volume to cover the surface of the plates at 37 °C until cells detach (time varies for different cell lines).
2. Collect cells by centrifugation at 300 rcf for 5 min. Wash cells two more times in PBS and collect by centrifugation.
3. Estimate the packed cell volume approximately from the graduations marked on the 1.8 ml tubes or 15 ml conical tubes.
   Note: Cells from culture can be snap-frozen in liquid nitrogen and stored at -80 °C indefinitely at this stage.

2. Isolation of Nuclei from Intact Cells

1. Thaw cells on ice.
2. Thaw nuclear isolation buffer (NIB, Table 1).
3. Prepare approximately 5 ml NIB buffer (Table 1) for every 100 µl packed cell volume. For every 1 ml NIB buffer, add protease inhibitors and stabilizing agents as follows: 1 µl of 1 M DTT, 2.5 µl of 200 mM AEBSF, 2 µl of 2.5 µM microcystin and 2 µl of 5 M sodium butyrate. NIB with inhibitors will be referred to as NIB from this point forward.
   Note: If histone phosphorylation is studied, include EDTA free protease and phosphatase inhibitor cocktail.
4. Remove a fifth volume of NIB buffer thus prepared and add NP-40 Alternative (Table 1) to a final concentration of 0.2% . The remaining four-fifth volume will be used for washes.
5. Wash cell pellet in 1:10 cell pellet to NIB without NP-40 Alternative ratio (v/v). Remove supernatant by centrifugation at 700 rcf for 5 min.
6. Lyse the cell pellet by placing it on ice and adding 1:10 cell pellet to NIB with 0.2% NP-40 Alternative (v/v).
7. If extracting from tissue samples, homogenize using mortar and pestle or dounce homogenizers. Cultured cells can be homogenized by gentle pipetting.
8. Incubate homogenized cells on ice for 5 – 10 min. The cells will lyse and release the nuclei.
9. Centrifuge at 1,000 rcf for 5 – 10 min at 4 °C. The pellet contains mostly cell nuclei, while the supernatant contains mostly cytoplasmic components. Save the cytoplasmic fraction if desired.
10. Wash the nuclei pellet by gently resuspending it in 1:10 (v/v) NIB without NP-40 Alternative.
    Note: This wash step is solely to remove traces of detergents prior to extracting histones from nuclei.
11. Centrifuge at 1,000 rcf for 5 min at 4 °C and remove supernatant.
12. Repeat Step 2.10 – 2.11 at least two times to completely remove NP-40 Alternative. Removal of NP-40 Alternative is evident as gentle pipetting during the washing step no longer forms bubbles.
13. For histone extraction from tissues:
    1. Rinse fresh or thawed frozen tissue in ice-cold NIB.
    2. Transfer tissue to a petri dish placed on ice with NIB, just enough to keep the tissue wet.
    3. Dice into smallest pieces (< 1 mm) with razor blade to increase the surface contact for nuclei isolation.
    4. Transfer minced tissue to a pre-chilled homogenizer and wash in NIB by pipetting up and down.
    5. Remove buffer by centrifuging at 300 rcf for 5 min.
    6. Add NIB containing NP-40 Alternative to the cells in a cells:buffer ratio of 1:10 (v/v) and homogenize by 5 – 10 strokes.
    7. Check for cell lysis and repeat homogenization as needed. A good indicator that cells have been lysed is the reduction of the pellet volume. The pellet should contain only nuclei.
    8. Centrifuge at 700 rcf for 5 min and save pellet. This pellet can be extracted 1 – 2 more times in 1:10 (v/v) of NIB containing NP-40 Alternative; at this stage, the histones are extracted out of chromatin and the pellet has shrunken considerably.
    9. Wash two times with 2 – 3 ml of NIB without NP40 Alternative to remove traces of the detergent.
       Note: Interim stopping point: Sample can be resuspended in the minimum volume of NIB + 5% glycerol, and stored at -80 °C.

3. Extraction and Purification of Histones from Nuclei

Note: Histones are very rich in basic amino acid residues, allowing them to tightly interact with the phosphoric acid backbone of DNA. Histones are among the most basic proteins in the nucleus, thus allowing them to be extracted in ice-cold sulfuric acid (0.2 M H₂SO₄) with minimal contamination from non-histone proteins, which precipitate in strong acid. Highly concentrated TCA (to a final concentration of 33%) can then be used to precipitate histones from the sulfuric acid. TCA is stored as 100% in brown bottle at 4 °C.

1. Resuspend cell nuclei in 1:5 (v/v) chilled 0.2 M H₂SO₄ (Table 1) by gentle pipetting.
2. Incubate the sample with constant rotation or gentle shaking for 2 – 4 hr at 4 °C. Typically, for samples with more than 500 µl cell pellet, a 2 hr extraction is sufficient to extract histones; longer incubation may result in extraction of other basic proteins. For smaller nuclear pellets (< 200 µl), 4 hr extraction provides a better yield.
3. Centrifuge at 3,400 rcf at 4 °C for 5 min.
4. Transfer the supernatant to a new tube.
5. Repeat Steps 3.3 – 3.4 to remove any insoluble material.
6. To precipitate the histones, add chilled 100% TCA (Table 1) to the collected supernatant (now containing histones) in the ratio of 1:3 (v/v), in order to obtain a final TCA concentration of 33%. Mix by inverting the tube a few times.
   Note: The samples will turn cloudy on addition of TCA, indicating the presence of histones.
7. Incubate the mixture on ice for at least 1 hr. For smaller starting pellet sizes, overnight precipitation is recommended.
8. Centrifuge at 3,400 rcf for 5 min. The histones coat the sides of the tubes and also deposit at the bottom. A white insoluble pellet also forms at the very bottom of the tube, which mostly contains non-histone proteins and other biomolecules. Remove supernatant by aspiration, carefully without scraping the sides or the pellet.
9. By using a glass Pasteur pipette, rinse the tube with ice-cold acetone + 0.1% HCl (Table 1) so as to cover the precipitated proteins coating the sides and bottom.
10. Centrifuge at 3,400 rcf for 2 min and aspirate supernatant, carefully without scraping the sides or the pellet.
11. Repeat Steps 3.9 – 3.10 using 100% ice-cold acetone.
12. Dry pellet with air flow or with a vacuum centrifuge, or just by leaving the tube open. Acetone evaporates quickly.
13. Dissolve the histones with ddH₂O (double distilled water) in minimum volume possible to dissolve the white layer completely. Histones are readily soluble in water. For pellets in a 1.5 ml microcentrifuge tube, 100 µl ddH₂O is usually enough to collect histones.
14. Centrifuge at 3,400 rcf for 2 min and transfer the supernatant to a new tube.

4. Estimation of Protein Concentration and Purity

1. For measuring protein concentration, use BCA, Bradford protein assay or amino acid analysis (AAA). Do not use techniques that adopt absorbance at 280 nm, as histones are poor in aromatic amino acid residues.
2. Verify the purity of extracted histones by SDS-PAGE analysis with a 15% acrylamide gel and Coomassie staining (optional).
3. If high purity single histone variants are desired, continue to HPLC-UV fractionation of histone variants (section 5). If not, skip directly to sample preparation for bottom-up histone PTM analysis (section 6).

5. Separation of Histone Variants by Reversed-phase HPLC (Optional)

Note: High purity histone variants can be obtained by fractionating the crude histone mixture using reversed-phase HPLC coupled to a UV detector. These purified histones are useful for studies that require higher sensitivity and purity. However, for standard histone PTM characterization, this step can be skipped because the analysis is sufficiently sensitive and exhaustive. Fractionation of intact histone variants ideally requires at least 100 – 300 µg of starting material.

1. Connect an appropriate C₁₈ 5 µm column to an HPLC depending on the starting histone concentration: with about 100 µg of histones, use 2.1 mm x 250 mm column with a flow-rate of 0.2 ml/min; with about 300 µg histones, use 4.6 x 250 mm column with a flow-rate of 0.8 ml/min. Prepare Buffer A and B using dedicated glassware as follows:
   1. Prepare Buffer A: 5% HPLC grade acetonitrile, 0.1% TFA in HPLC grade water.
   2. Prepare Buffer B: 95% HPLC grade acetonitrile, 0.1% TFA in HPLC grade water.
2. Connect the column to a UV detector, and set the absorbance to 210 – 220 nm.
3. Acidify the histone sample dissolved in water with 100% TFA to achieve a final concentration of 0.1-1% TFA.
4. Equilibrate the column with 100% buffer A for at least 15 min at the recommended flow-rate, which corresponds approximately to three column volumes. Use this signal to set the zero-absorbance level of the UV detector.
5. Prepare appropriately sized tubes to collect fractions either manually or in an automatic sample collector.
6. Inject sample at a concentration of approximately 1 µg/µl or higher. Samples dissolved in larger volumes might alter the equilibration of the column during loading and lead to lower retention.
7. Run the gradient, programmed as follows: from 0 to 30% B in 1 min, 30 to 60% B in 90 min, and 60 to 90% B in 1 min.
8. Collect fractions (example chromatogram shown in Figure 2) in 1 min intervals using an automatic fraction collector. Collect fractions in appropriate size tubes to contain the entire volume.
9. Dry down fractionated samples in a vacuum concentrator.
   Note: Interim stopping point: Dried histone fractions can be stored at room temperature for short periods (1 – 2 days) or in -80 °C freezer for long-term periods.

6. Chemical Derivatization of Histones Using Propionic Anhydride for Bottom-up Analysis

1. Dissolve histone samples in 40 μl of 50 mM NH₄HCO₃, pH 8.0 (recommended amount: 50 – 100 μg). If samples were in pure ddH₂O, add concentrated NH₄HCO₃ to make up 50 mM, pH 8.0.
2. Wet a P10 pipette tip into the sample to check the pH using pH indicator strips without sample losses. NH₄OH and formic acid can be used to adjust the pH to 8.0.
   Note: The following part of the protocol (steps 6.3 – 6.7) should be done in batches of maximum three to four samples, in order to keep propionic anhydride reactive.
3. Use fume hood for the subsequent steps where propionic anhydride is used. Prepare fresh propionylation reagent by mixing propionic anhydride with acetonitrile in the ratio 1:3 (v/v). Add propionylation reagent to sample in 1:4 (v/v). For 40 µl histones, add 10 µl propionylation reagent.
   Note: It is possible to observe white debris at this step. However, this mostly contains salts and propionic acid, and thus no specific action needs to be taken.
4. Quickly add NH₄OH to re-establish pH 8.0 to the solution. Note: Propionic anhydride reacting with the free amines of the peptides produces propionic acid that decreases pH. Usually, adding NH₄OH to the sample with a ratio of 1:5 (v/v) is appropriate to re-establish pH 8.0; e.g., 8 µl of NH₄OH to 40 µl of sample.
5. Mix immediately by vortexing.
6. Check pH with the same procedure as Step 6.2.
   Caution: When pH is greater than 10.0, labeling of other amino acid residues with higher pKa is possible.
7. Incubate samples at room temperature for 15 min.
8. Repeat Steps 6.3 – 6.7, strictly performing the reaction for no more than 3 or 4 samples per batch of propionylation reagent.
9. Dry samples down to 10 – 20 µl in a vacuum concentrator. This evaporates unreacted propionic anhydride, acetonitrile, acetic acid and ammonia gas released from NH₄OH. If samples dry out completely, no significant sample losses occur.
   Note: Isopropanol can be used instead of acetonitrile. However, acetonitrile has lower surface tension and thus more rapid evaporation.
10. Resuspend or dilute samples with ddH₂O until 40 µl of final volume is achieved.
11. Repeat Steps 6.2 – 6.9. A double round of histone propionylation ensures > 95% of reaction completion.
12. Fill propionic anhydride bottle with argon gas so as to prevent formation of acetic acid in contact with moisture in the bottle.
    Note: Interim stopping point: Sample can be stored at -80 °C reconstituted in ddH₂O or dried.

7. Proteolytic Digestion with Trypsin

1. Resuspend histones in 50 mM NH₄HCO₃ to achieve an optimal concentration of 1 μg/μl or higher. More diluted samples lead to lower trypsin efficiency.
   Note: The histones at this step need to be at pH 8.0 If it still acidic, then add NH₄HCO₃ salt to sample by using pipet tip.
2. Add trypsin to histone samples at a 1:10 ratio (wt/wt).
3. Incubate at 37 °C for 6 – 8 hr.
4. Stop the digestion by freezing in -80 °C.
5. Dry down the sample to 10 – 20 µl in a vacuum concentrator.
   Note: Interim stopping point: Sample can be stored at -80 °C.

8. Propionylation of Histone Peptides at N-termini

Note: This section describes the derivatization of peptide N-termini generated from the trypsin digest. Such procedure improves HPLC retention of the shortest peptides (e.g., amino acid 3 – 8 of histone H3), as the propionyl group increases peptide hydrophobicity.

1. Resuspend samples in 30 µl of 100 mM NH₄HCO_3.
2. Repeat Steps 6.1 – 6.9.
   Note: It is normal that drying of samples in vacuum takes a longer time at this step.
3. Resuspend or dilute samples with 50 – 100 µl ddH₂O + either 0.1% TFA or 0.5% acetic acid. Note: Acetic acid is recommended for long storages, as TFA facilitates methionine oxidation in the long term. On the other hand, TFA is recommended if stage-tipping (section 9) is performed the same day, as TFA assists a better chromatographic retention.
   Note: Interim stopping point: Sample can be stored at -80 °C.

9. Sample Desalting with Stage-tips

Note: At this stage, there is salt present in the sample. Salts impede HPLC-MS analysis because they ionize during electrospray, suppressing the signal from peptides. Salts can also form ionic adducts on peptides, reducing the signal intensity for the non-adducted peptide. As the adducted peptide will have a different mass, the peptide will not be properly identified or quantified.

1. By using a P1000 pipette tip, punch a disk of C₁₈ material from a solid phase extraction disk. Push the minidisk out of the P1000 tip by using a fused silica capillary and deposit the minidisk to the bottom of a P100 / 200 pipette tip. Ensure that the disk is securely wedged at the bottom of the tip (Figure 3).
   Note: The P1000 tip has a rather small hole to punch the C₁₈ disk. It is appropriate to cut the last centimeter of the tip in order to have a hole with larger diameter.
2. Use two C₁₈ punches in the same P100/P200 tip if desalting over 25 µg of sample.
3. Use a centrifuge adaptor to hold stage-tips in place in 1.5 ml or 2 ml microcentrifuge tubes. Use slow (300 – 400 rcf) rotation; the solvents normally pass through the resin in less than a minute.
4. Flush the resin by spinning with 50 µl of 100% acetonitrile to activate the C₁₈ material and remove potential contaminations.
5. Equilibrate disk by flushing 80 µl of 0.1% TFA by slow centrifugation.
6. Acidify the sample to pH 4.0 or lower with acetic acid. Check the pH with pH strips to minimize sample loss. Load sample onto the disk by slow centrifugation.
7. Wash sample by flushing 70 – 80 µl of 0.1% TFA by slow centrifugation.
8. Elute sample by flushing 70 µl 75% acetonitrile and 0.5% acetic acid by slow centrifugation. Collect the sample in a 1.5 ml tube.
9. Dry sample in a vacuum concentrator.
   Note: Interim stopping point: Sample can be stored at -80 °C.

10. Analysis of Histone Peptides

Note: The nLC-MS platform should be set up as done in traditional peptide analysis. The use of 200 – 300 nl flow column (75 µm ID analytical column, C₁₈ particles) is recommended, as they are an excellent compromise between sensitivity and stability. The MS acquisition method can be either a combination of data-dependent acquisition (DDA) with targeted scans¹⁹ or a data-independent acquisition (DIA)^20,21, both described in Representative Results and Figure 4.

1. Prepare HPLC buffers — A: 0.1% formic acid in HPLC-grade water; B: 0.1% formic acid in HPLC-grade acetonitrile.
2. Program the HPLC method as follows: from 0 to 30% buffer B in 30 min, from 30 to 100% B for the next 5 min and at isocratic 100% B for 8 min. If the HPLC is not programmed for automated column equilibration before sample loading, then include the following: gradient from 100 to 0% B in 1 min and isocratic flow at 0% B for 10 min. Set the flow rate of the analysis to 250 – 300 nl/min.
3. Program the MS acquisition method to perform either DDA combined with targeted scans¹⁹ or DIA ^20,21 (Figure 4). Make sure that the MS duty cycle allows one full MS scan every ~ 2 sec, in order to have enough data points to across the chromatographic peak, which enables more accurate quantification. Note: With C₁₈ chromatography, the average baseline peak width is about 30 sec for the gradient described above.
4. Load approximately 1 µg of sample onto the HPLC column.
5. Run the HPLC-MS/MS method as programmed.
   Note: In the protocol we do not recommend specifics of columns, MS instruments or MS parameter details, as any optimal setup that an individual proteomics lab developed will be suitable for the method. Proteomics laboratories should use their optimized setup, since histone peptides separate as traditional peptides.

11. Data Analysis

1. Import the MS raw files into software to perform peak area integration. Note: EpiProfile²² is recommended, as it is optimized for histone peptides; by using the retention time knowledge of chromatographic elution it performs reliable peak area extraction of known histone peptides. Alternatively, Skyline²³ is another ideal software for the purpose.
   1. Calculate the relative abundance of a given peptide by dividing its area by the total area of that peptide in all of its modified forms. Note: In case of discovery analysis Mascot is recommended to identify spectra of modified histone peptides. The performance of this tool has been described recently²⁴. All other database search engines for proteomics are also usable, but upon our tests they provided lower performance.