Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

NOTE: Histone samples were extracted using a method adapted from Bhanu et al. (2020)¹².

1. Sample preparation

1. Harvesting cultured cells
   1. When cells are 80% confluent, ensure they are viable using trypan blue exclusion.
      NOTE: A HeLa S3 cell line was used for these experiments, but this method can be applied to any cultured cells.
   2. Aspirate the media, then apply 5 mL of 1x phosphate-buffered saline (PBS) to each plate.
   3. Swirl the plate(s) to rinse all residual media, then aspirate PBS and apply 5 mL of 1x PBS.
   4. Gently separate the cells from the plate by scraping them with a disposable cell lifter.
   5. Transfer each cell suspension to a 15 mL conical tube.
   6. Pellet the cells by centrifuging at 800 x g for 5 min.
   7. Aspirate the PBS from the cell pellet.
   8. Proceed to histone extraction.
      NOTE: Flash-freeze the cell pellet in liquid nitrogen if it cannot be processed immediately. Store the pellets at -80 °C until ready to proceed.
2. Histone extraction
   1. Estimate the volume of each cell pellet and mark the meniscus with a permanent marker.
   2. Prepare enough nuclear isolation buffer (NIB; 15 mM Tris-HCl (pH 7.5), 15 mM NaCl, 60 mM KCl, 5 mM MgCl₂, 1 mM CaCl_2, and 250 mM sucrose) for all the samples. Alternatively, if many samples need to be processed over time, make the buffer in bulk and store at 2-8 °C for up to 6 months, or aliquot and freeze at -15 °C to -25 °C indefinitely by thawing only the amount necessary for each extraction.
      NOTE: The buffer should remain clear during storage. If the buffer takes on a cloudy or otherwise abnormal appearance at any time, discard and prepare fresh buffer.
   3. Prepare 50 times the volume of the cell pellets of wash buffer and add inhibitors as follows (approximately 10 mL of wash buffer per 2 samples).
      1. To prepare 10 mL of wash buffer, mix 10 mL of NIB, 30 µL of 200 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride [AEBSF], 10 µL of 1 M dithiothreitol [DTT], 20 µL of 5 µM microcystin, 20 µL of 5 M sodium butyrate.
   4. Remove 1/5 of the wash buffer to prepare the lysis buffer (1/5 volume from wash buffer, 0.3% NP-40 or NP-40 alternative).
      NOTE: Do not use Triton-X 100 instead of NP-40 or NP-40 alternative, as it may be too abrasive for certain cell types.
   5. Wash the cell pellet thoroughly by suspending it in 5 columns of wash buffer and centrifuging at 800 x g for 5 min at 4 °C. Complete this step twice, aspirating and discarding the supernatant between washes.
   6. Ensure the volume of the cell pellet is still marked with a permanent marker. Resuspend in 10 volumes of lysis buffer.
   7. Pipette-mix each pellet thoroughly to resuspend, then incubate for 15 min on ice.
   8. After 15 min, centrifuge at 800 x g for 5 min at 4 °C.
   9. Aspirate and discard the supernatant.
      NOTE: The pellet should reduce to ≤ ¹/₂ the original pellet size (as indicated by the marker line). If the pellet has not reduced sufficiently, repeat the lysis procedure and include a gentle homogenization step using a pestle to break open the cells.
   10. Once lysis is complete, resuspend the pellet in 500 µL of wash buffer, then centrifuge at 800 x g for 5 min at 4 °C. Aspirate and discard the supernatant, then repeat the wash step once more to remove all traces of NP-40.
       NOTE: At this point, the pellet consists of chromatin, which contains histones.
   11. Resuspend the pellet in 5 volumes (of the original cell pellet size) of 0.4 N H₂SO₄.
   12. Incubate for 2 h in a cold room or refrigerator using an agitator.
   13. After 2 h, centrifuge the sample(s) at 3400 x g for 5 min at 4 °C. Do not discard the supernatant.
   14. Transfer the supernatant to new tubes, and spike 100% trichloroacetic acid (TCA) to 1/3 the volume of the contents (the final TCA concentration will be approximately 20%).
   15. Gently invert the tube and observe that the clear, colorless solution turns white and/or cloudy, indicating protein precipitation.
       NOTE: For solutions with low histone concentrations, the protein precipitation may not be immediately noticeable, but the precipitate should be visible after the overnight incubation.
   16. Incubate without disturbance overnight (12-18 h) at 4 °C to completely precipitate the histone proteins.
   17. The following day, centrifuge at 3400 x g for 5 min at 4 °C.
   18. Aspirate the supernatant, being careful not to touch the sides of the tube with the pipette tip. At this stage, the histones are deposited primarily as a film around the sides of the tube(s).
   19. Add 500 µL of ice-cold acetone + 0.1% HCl (acid acetone) to each tube, using a glass Pasteur pipette, then gently invert the tube(s) several times. Arrange the samples in order (1, 2, 3, etc.) while doing this, as any errant acetone may remove the markings on the tubes. Centrifuge at 3400 x g for 5 min at 4 °C and gently decant the supernatant.
   20. Repeat this rinsing step with 500 µL of ice-cold 100% acetone, also with a glass Pasteur pipette. Centrifuge at 3400 x g for 5 min at 4 °C and gently decant the supernatant.
   21. Leave the tubes open to dry at room temperature until the remaining acetone has evaporated.
   22. When dry, add 100 µL of mass spectrometry (MS)-grade water to each tube. Use this droplet to swab all sides of the container to resuspend the entire histone film. Do this by pipetting the droplet onto the side of the tube and rotating it while pipetting up and down or dispensing half of the 100 µL and using the tip to stir it all around. A combination of both methods works best. Histones are readily soluble in water and will be in the solution.
   23. After resuspending all samples, if there is any remaining white solid, sonicate in a bath at room temperature for 5 min.
   24. Centrifuge at 800 x g for 5 min at 4 °C. Transfer the clear solution to fresh tubes. Discard any remaining insoluble pellet.
   25. Run an SDS-PAGE under reducing conditions to verify that the extraction is clean.
       NOTE: Gels can be run using any appropriate concentration of polyacrylamide as long as it can differentiate proteins in the range of 10-20 kDa. See the Table of Materials for the gels used in this protocol.
   26. Perform a protein concentration assay (i.e., Bradford or BCA) to determine the total protein concentration.
3. Chemical derivatization (propionylation) of the lysine residues
   1. Transfer 20 µg of histones (determined by the protein assay) to a clean tube. Dry this sample down to <5 µL using a vacuum concentrator, then resuspend using 20 µL of 100 mM ammonium bicarbonate (NH₄CO₃) (~1 µg/µL solution). Adjust the pH to ~8 using ammonium hydroxide if needed.
      CAUTION: Do not use ammonium hydroxide (NH₄OH) to resuspend, only to adjust the pH if necessary. Otherwise, proteins will denature and precipitate.
      NOTE: To check the pH with minimal sample loss, use a pipette tip to dip in the sample and dab onto a pH strip. This testing procedure will be useful throughout the remaining sample preparation steps.
   2. Prepare the propionylation reagent by adding propionic anhydride to acetonitrile (ACN) in a 1:3 (v/v) ratio (i.e., to make 40 µL of reagent, combine 10 µL of propionic anhydride with 30 µL of ACN).
      NOTE: Traditionally, methanol or isopropanol have been used in preparation of a propionylation reagent. As propionylation is an amide formation reaction, a non-protonic solvent, like acetonitrile, is required to prevent unwanted side products and reactions, such as methyl propionyl ester, which results from using methanol. Only prepare enough propionylation reagent for up to 4 samples at a time so the reagent remains fresh. Use the reagent within 1-2 min of preparation. As the reagent sits, the propionic anhydride will react with any ambient moisture, and acetic acid will begin to form, which can change the effectiveness of the reagent and will change the pH of the histone solution once the reagent is added.
   3. Add propionylation reagent to each sample in a 1:4 (v/v) (i.e., for 20 µL of histones, add 5 µL of propionylation reagent).
   4. Quickly add 1:5 (v/v) NH₄OH (i.e., add 4 µL for 20 µL of the histone solution) to re-establish the pH of the solution to ~8. If pH is still too low, add 1-2 µL of NH₄OH at a time until a pH of 8 is achieved. Typically, a 1:5 (v/v) ratio is adequate.
   5. Incubate the samples at room temperature for 15 min without disturbance.
   6. Repeat the propionylation reaction for no more than 3-4 samples per batch of propionylation reagent to ensure minimal acid formation.
   7. Repeat the propionylation procedure steps 1.3.2-1.3.5. A second round of propionylation ensures that >95% of available lysines are derivatized.
   8. Dry the samples down to <5 µL using a vacuum concentrator. This will evaporate any unreacted propionylation reagent, acid products, and ammonia gas released from the NH₄OH. If the samples dry out completely, this is fine, as no significant sample losses occur.
      NOTE: Displace air in the propionic anhydride bottle with argon gas prior to storage to prevent the formation of acetic acid due to contact with ambient moisture remaining in the bottle.
4. Proteolytic digestion with trypsin
   1. Resuspend histones in 100 mM NH₄HCO₃ to achieve a volume of 20 µL, achieving an optimal concentration of 1 µg/µL.
      NOTE: Sample solutions with concentrations lower than 1 µg/µL will result in decreased Trypsin efficiency.
   2. Add trypsin to histone samples at a 1:10 ratio (wt/wt) (i.e., add 2 µL of 1 µg/µL solution of trypsin to 20 µg of histones).
   3. Incubate reactions at 37 °C for 6-8 h. Alternatively, incubate overnight (12-18 h) at room temperature.
   4. Stop the digestion by freezing at -80 °C for at least 1 h.
      NOTE: Do not use acid to quench the digestion reaction, as this will cause an unwanted drop in pH at this point in the procedure. The sample can be stored at -80 °C until ready to proceed (Interim stopping point).
5. Chemical derivatization (propionylation) of peptide N-terminals
   1. Dry the samples to <5 µL using a vacuum concentrator.
   2. Resuspend the samples up to 20 µL (1 µg/µL) using 100 mM NH₄HCO₃.
   3. Repeat propionylation as before (step 1.3).
      NOTE: It is normal that the samples take longer to dry at this step due to a higher aqueous: organic phase ratio.
6. Sample desalting with stage-tips
   1. Resuspend or dilute samples with 50 µL of MS-grade water + 0.1% TFA.
   2. Using an 11-G sample corer, punch 5 disks of C₁₈ material from a solid phase extraction disk (punch all 5 disks before transferring to the pipette tip). Insert and ensure the disks are securely and evenly wedged at the bottom of a 200 µL pipette tip (Figure 1).
      NOTE: Use 15-G corer if desalting over 25 µg of sample through a single stage-tip.
   3. Use a centrifuge adapter to hold the stage-tips in place in 1.5 mL or 2 mL microcentrifuge tubes.
      NOTE: For the following centrifugation steps, use slow (400-500 x g) revolution at 4 °C, for 1-2 min at a time; the solvents normally pass through the resin in less than 1 min, depending on how tightly the C₁₈ material is packed into the tips.
   4. Rinse the resin by centrifuging with 50 µL of 100% acetonitrile to activate the C₁₈ material and remove potential contaminants.
      NOTE: It may be easier to load solutions onto the stage-tips using gel-loading pipette tips. Once the C₁₈ material has been activated, it is important not to allow the resin to dry out for the duration of the desalting procedure.
   5. Equilibrate the disk material with 80 µL of MS-grade water + 0.1% TFA by centrifugation.
   6. Acidify the sample to pH 4 or lower using glacial acetic acid. Check the pH with pH strips as before to minimize sample loss.
   7. Load the entirety of the sample onto the resin disk by slow centrifugation.
   8. Wash the sample with 80 µL of MS-grade water + 0.1% TFA by centrifugation.
   9. Elute the sample into a clean 1.5 mL tube by flushing 70 µL of 75% acetonitrile and 0.5% acetic acid by slow centrifugation. Additional centrifugation time may be used to ensure the full sample volume is eluted from the stage-tip. It is okay if the resin dries out with the additional centrifugation time, as it is no longer needed past the elution of the sample.
   10. Dry each sample completely in a vacuum concentrator.
       ​NOTE: Sample(s) can be stored at -80 °C until ready to proceed (Interim stopping point).
   11. For LC-MS/MS analysis, reconstitute the samples in a volume of Solvent A (0.1% formic acid) from the liquid chromatography (LC) protocol that gives the final concentration of 0.4 µg/µL (i.e., dissolve 20 µg of histones in 50 µL of Solvent A).

2. TIMS software interface

1. Select the Instrument tab and switch to Operate (verify that the instrument name becomes highlighted in green) (Figure 2).
2. Verify TIMS parameters (Figure 2).
3. Verify MS Settings (scan begin, scan end, ion polarity, scan mode) (Figure 2).
4. Verify TIMS settings (mode, mobility start, mobility end, ramp time, accumulation time, duty cycle, ramp rate, MS rate, MS averaging, and autocalibration) (Figure 2).
5. Go to the Source tab and activate the syringe option (Hamilton 500 µL) only for the TuneMix calibration step (Figure 3)¹³.
6. Go to the Calibration tab, click m/z, select Calibration Mode, and choose the mode (Enhanced Q, generally), zoom (+0.01%) STD Dev (0.24), and click Calibrate; when a score of 100% is achieved, accept (Figure 4).
7. Go to the mobility tab and repeat the calibration process (Linear Mode, generally), detection range (+5%), width (0.1 Da), STD Dev (0.1855), then click calibrate; when you get a score ≥ 98.5%, accept (Figure 5).
8. Go to the method and select the method to be used; for this example, Proteomic_/2023-01-19-CF/20230119-Hela Control histone_prep_pasefDIA_1-24_1_451.d/451.m-TimsControl was selected (Figure 5).

3. LC-TIMS-PASEF-ToF MS/MS

1. Use the typical nESI operating conditions: 4500 V capillary voltage, 800 V endplate offset, 3.0 bar nebulizer pressure, 10.0 L/min dry gas, 200 °C dry heater, and 50 µL/min injection flow rate.
2. Use the typical MS settings: 6 eV collision energy, 1200 Vpp collision RF, 75 µs transfer time, 5 µs prepulse storage.
3. Determine the drift gas flow using the pressure difference from the entrance funnel P1 and the exit funnel P2. Parallel accumulation-serial fragmentation (PASEF) occurs in the TIMS cell, accumulating all precursor ions simultaneously rather than individually. Precursor ions are then released in narrow ion peaks versus the normally much wider peaks (about 50 times shorter), increasing the signal-to-noise ratio while still separating co-eluting peptides via mobility¹⁴.
4. Develop an LC-TIMS-ToF MS/MS method for analyzing proteolytic histone peptides. Couple a high-performance liquid chromatograph (HPLC) fitted with a C₁₈ (300 Å, 5 µm, 4.6 mm x 250 mm) column with a commercial TIMS-TOF MS instrument with proprietary PASEF technology.
   NOTE: This column size was determined to provide good separation at both high and low pH for peptide mixtures, based on previously published works^15,16,17.
   1. Set the injection volume to 20 µL (8 µg) of sample and a 0.4 mL/min flow rate.
   2. Run a 60-min, non-linear LC gradient using water with 0.1% formic acid (Solvent A) and acetonitrile with 0.1% formic acid (Solvent B). Set the gradient: 10% B for 2.7 min, then to 20% B in 5.3 min, 28% B in 4 min, 35% B in another 18 min, to 40% B in 13 min, and 100% B in another 2 min. After holding 100% B for 5 min, lower the concentration to 10% B in 5 min and hold for the final 5 min.
5. Verify sample elution from the HPLC into the TIMS-TOF via nano-electrospray ionization (nESI) in positive ionization mode.

4. Data analysis

1. Identify the peptide sequences and modification sites.
   1. Prepare a theoretical list of peptides using ProteinProspector [https://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msdigest] under the MS-digest tool.
      1. Perform a theoretical digest while taking into consideration the conditions of the digest (enzyme used), types of PTMs being searched for (e.g., mono-, di-, or trimethylation), the size range of peptides being searched for, as well as mass detection range and the potential number of missed cleavages.
2. Manually analyze the acquired data based on theoretical peptides (Figure 6)¹².
   1. Search for the masses at several charge states (+1 to +4) for each theoretical peptide are searched for.
   2. Following the initial identification of each m/z, select the peak and confirm the MS/MS using a theoretical list of fragmentation ions based on the peptide sequence, including PTMs.
      NOTE: If the mobility of the identified peptide was known previously, this is also confirmed.
3. Calculate the relative abundances of various PTMs and report each modification as a percentage of the specified peptide sequence.
   1. The relative abundance of each detected PTM is calculated using the following equation:
      Relative abundance = Area of PTM/Total area of unmodified and PTMs for a given peptide