Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

1. Histone extraction

1. Perform histone extraction from at least 1 x 10⁶ cells, which are 80% confluent. For this experiment, we used MCF-10A cell lines from ATCC. After plating, wash cells thrice with 1 mL of phosphate-buffered saline (PBS) 1x. Perform all the steps on ice.
2. Prepare a cell lysis buffer containing 10 mM Tris-HCl pH 8.0, 1 mM KCl, and 1.5 mM MgCl₂. Add 1 mM dithioerythritol and protease and phosphatase inhibitors just before use.
3. Add 800 µL of cell lysis buffer to the cell-containing Petri dish, scrape, and collect the cells in a 1.5 mL tube, then incubate on a rotator at 300 rpm, 4 °C for 1 h.
4. Centrifugate at 10,000 x g, 4 °C, for 10 min in a precooled microcentrifuge with a rotator for 1.5 mL tubes.
5. Wash the pellet with 400 µL of cell lysis buffer and centrifuge at 10,000 x g, 4 °C, for 10 min. Discard the supernatant. Repeat the washing step 3x.
   NOTE: Use a precooled microcentrifuge (4 °C) with a rotator for 1.5 ml tubes for all the steps. Handle the pellet carefully to avoid loss of material.
6. Resuspend the pellet in 640 µL of 0.2 M H₂SO₄ and incubate on a rotator at 300 rpm, at 4 °C, for 16 h.
   CAUTION: Sulfuric acid is a hazardous chemical; please use it under a chemical fume hood.
7. Centrifuge at 16,000 x g, 4 °C, for 10 min. Transfer the supernatant to a fresh tube and store it until the following steps. Discard the nuclear debris.
8. Precipitate histones by adding 212 µL of Trichloroacetic acid, drop by drop (the final concentration of TCA is 33%). Mix the solution by inverting the tube 10x. Incubate on ice, in the dark, for 16 h.
   CAUTION: TCA is a hazardous chemical; use under a chemical fume hood.
9. Pellet histones by spinning in a precooled centrifuge at 16,000 x g for 10 min at 4 °C. Carefully remove the supernatant and wash the pellet 3x with 200 µL of cold acetone.
10. Resuspend the pellet with cold acetone, then sonicate high power 2x with 3 cycles of 30 s on/off, alternating with a 10 min pause on ice.
    NOTE: The procedure was done using an ultrasonicator. This step is essential to eliminate histone-wrapped DNA.
11. Centrifuge at 16,000 x g for 10 min, discard acetone, and resuspend the sample in 100 µL of double-distilled water. Determine protein concentration using the Bradford assay method, according to the manufacturer's instructions³³.
    NOTE: After centrifugation, the pellet sometimes appears white or translucent. A white and opaque precipitate could indicate the presence of salt contaminants that might impair the 2D separation. If so, repeat the acetone wash steps until the pellet becomes translucid.
12. Lyophilize 70 µg of histones, according to the manufacturer's instructions. Set the lyophilizer at 30 °C for 60 min. Check the tube volume and reiterate the step at 30 °C until the sample is completely lyophilized (generally, 90 min is necessary to completely lyophilize the sample). After 60 min, check the volume every 10 min since the process is sample-dependent.
    NOTE: If the amount of histone proteins is low, start from 2 x 10⁶ cells. In this case, use the same volume of reagents used for 1 x 10⁶ cells. Check the purity of all the chemicals used during the procedure; all the reagents must be HPLC grade.

2. Mini 2D gel triton-acid-urea (TAU) electrophoresis

1. Prepare a mini-TAU gel for a mini vertical polyacrylamide gel electrophoresis system as described below.
   1. Prepare separating gel: 6 M Urea, 15% acrylamide/bisacrylamide (29:1), 5% glacial acid acetic, 0.25% Triton X-100 (v/v), 0.4% TEMED (v/v), 0.1% APS (p/v). To prepare the solution, dissolve urea in acrylamide/bis and 1 mL of ultrapure water with gentle agitation at room temperature; add the other components and adjust the volume. For a mini-TAU gel, prepare 10 mL of solution. Fill the preset gel plates with the solution, avoiding the formation of air bubbles just 1.5 cm from the top. Cover the solution with 1 mL of isopropanol to level out the gel.
   2. Prepare stacking gel: 6 M Urea, 6% acrylamide/bisacrylamide (29:1), 5% glacial acid acetic, 0.25% Triton X-100 (v/v), 0.4% TEMED (v/v), 0.1% APS (p/v). To prepare the solution, dissolve urea in acrylamide/bis and 1 mL of ultrapure water; add the other components and adjust to volume. Fill gel plates with stacking solution, place a 10-well gel comb between plates, and insert it until the gel solution comes halfway up the teeth of the comb. Allow the gel to polymerize 16 h at 4 °C.
      NOTE: Carefully insert the comb to ensure the regular formation of wells.
2. Prepare 100 µL of TAU sample buffer by dissolving 6 M Urea, 5% glacial acid acetic, and double-distilled water. Dissolve the lyophilized sample prepared in 30 µL of TAU sample buffer.
3. Fill the mini cassette with TAU running buffer, 5% acetic acid final concentration. Rinse the wells of the TAU-gel with a syringe filled with running buffer and load the sample in the well using a micropipette.
4. Connect the electrophoretic chamber to the power supply, with the positive (red) electrode placed at the bottom. Pay attention, when IP > pH, proteins are positively charged, so the migration is from positive to negative electrode. Run the gel first at 30 V for 70 min, then at the constant voltage of 25 V for 17 h.
   NOTE: During this time, occasionally switch off the power supply and rinse the gel wells using a disposable syringe. Pay attention to removing any bubbles from the bottom surface of the gel using a syringe to avoid the loss of sample and the presence of a smearing in the resolved proteins.
5. Remove the gel from the gel plates and cut the line of interest with a clean scalpel. Carefully handle the gel to avoid gel breakage. Since the line of interest is colorless, load the sample in well number 1 to exactly identify and cut the lane of interest.
   NOTE: To check the separation on mini-TAU gel, prepare and run the sample in duplicate. One of these gels can be stained using Coomassie Brilliant Blue. The pattern of separation is reported in Figure 1A.
6. Wash the lane with 15 mL of 0.5 M Tris-HCl pH 8.8 for 15 min.
7. Prepare 15 mL of equilibration buffer 1 using 6 M Urea, 0.5 M Tris-HCl, pH 6.8, 30% glycerol, 20% SDS, and 2% Dithioerythritol. Prepare 15 mL of equilibration buffer 2, using 6 M Urea, 0.5 M Tris-HCl, pH 6.8, 30% glycerol, 20% SDS, and 2% Iodoacetamide. Prepare the equilibration buffer 1 and 2 just before use; store the equilibration buffer 2 in the dark.
8. Incubate the gel lane, under gentle agitation, in 10 mL of equilibration buffer 1 for 15 min, then decant. Incubate the gel lane, under gentle agitation, with 10 mL of equilibration buffer 2 for 15 min in the dark, then decant.
9. Prepare a resolving SDS 15% acrylamide gel by following Table 1. Set up the gel plate and fill it with the resolving gel solution. Fill the solution until it is 1.5 cm from the end of the long gel plate. Put 1 mL of isopropanol on the surface to level the gel and allow it to polymerize.
10. After gel polymerization, decant the isopropanol and dry the well with 3MM Chr Cellulose Chromatography Paper. Insert the resulting lane from step 8 into the gel well.
11. Seal the lane to the resolving gel with a sealing solution of 0.5% low melting point agarose in 1x Tris/glycine/SDS buffer and 5 µL of 0.003% bromophenol blue. Allow agarose gel polymerization for 30 min.
    NOTE: Avoid the formation of air bubbles during agarose sealing to prevent gel smearing.
12. Put the gel plates in the cassette and fill with 1x Tris/glycine/SDS buffer. Run the gel at 120 V until the bromophenol blue dye reaches the bottom. Gently remove the gel from the plates, discard the excess of agarose, and silver stain the gel using a procedure compatible with mass spectrometry or blot for immunological assay.

3. Silver staining ³²

1. Prepare the following reagents – Fixative: 50% methanol, 5% acetic acid, 45% deionized water; Wash solution: 50% methanol, 50% deionized water; Sensitizer: 0.02% Na₂S₂O₃ 5H₂O in deionized water; Silver reagent: 0.1% silver nitrate in cold (4°C) deionized water; Developer: 0.04% Formalin and 2% Na₂CO₃, Formaldehyde (37%) (add just before use) 0.4 mL deionized water and fill up to 1000 mL; Stop-reagent: 5% acetic acid; Gel storage solution: 1% acetic acid.
   NOTE: Since chemicals used for staining are hazardous, perform all the steps under a chemical fume hood.
2. Follow the procedure in Table 2 with the following consideration. Perform all incubations with gentle agitation. Prepare the stop reagent before starting the developing step; this will ensure gel development with the opportune intensity. During the developing step, spots will appear as yellow signals that will become light brown at the end of the incubation. The intensity of the different spots will depend on the amount of histone proteoforms. The gel is ready for spot excision and in-gel digestion (Figure 1B)

4. In-gel digestion

1. Prepare the following solutions: 50 mM ammonium bicarbonate (NH₄HCO3)/50% (v/v) acetonitrile; 10 mM dithiothreitol (DTT) in 50 mM NH₄HCO₃; 55 mM iodoacetamide (IAA) in 50 mM NH₄HCO₃; 0.1% TFA; 50 mM NH₄HCO₃, pH 8; HPLC grade Acetonitrile; 1 mL silicon tubes.
   NOTE: To circumvent human keratin contamination, from hair or skin, use clean gloves and work in a clean area.
2. Excise histone spots of interest from the stained gel using a clean scalpel and place them into a fresh silicon tube (1.5 mL). Take care to excise a single spot and put it in a single tube to avoid isoform cross-contamination.
3. Add to each gel spot 250 µL of 50 mM NH₄HCO₃/50% acetonitrile and incubate under gentle agitation in a thermomixer at room temperature. Decant the solution and repeat this step 3x. Gel pieces will appear transparent.
4. Dehydrate gel spots by adding acetonitrile (200 µL). At this stage the gel pieces will take an opaque-white color.
5. Decant acetonitrile and let air-dry for 10 min. The gel piece will appear dried in the tube. Reduction and alkylation are not essential since histone proteins have been reduced and alkylated during the equilibration step.
6. Rehydrate gel spots in 20 µL of trypsin solution (0.1 mM HCl) or a volume sufficient to cover the gel pieces. Place the tubes in the thermomixer and incubate, under gentle agitation, at 37 °C for a minimum of 4 h until 16 h.
7. Transfer the supernatant containing the tryptic peptides to a fresh tube. Add 50 µL of extraction solution (60% acetonitrile, 1% TFA) to the gel pieces and sonicate 2x in an ultrasonic water bath for 10 min at 4 °C.
8. Transfer the supernatant, containing additional tryptic peptides, to the fresh tube. Use a lyophilizer to dry the pooled extracted peptides. Set the lyophilizer at 30 °C and dry the solution; a lyophilized pellet will appear in the tube.
   NOTE: This step is volume-dependent; generally, 1 h is enough, but check the volume every 10 min until the sample is completely dried.
9. Add 15 µL of 0.1 % TFA to each tube and gently incubate in a thermomixer at 25 °C. Transfer the supernatant to the vial for LC-MS/MS analysis.

5. 2DTAU western blot

1. Prepare the transfer buffer, 250 mM Tris base, 92 mM glycine, and 10% methanol using double-distilled water.
2. Cut the nitrocellulose membrane and the filter paper to the dimensions of the gel.
3. Equilibrate the gel and soak the membrane, filter paper, and filter papers in the transfer buffer for 15 min. Use either Nitrocellulose or PVDF membranes. If using PVDF (Polyvinylidene difluoride) membranes, activate them by soaking in 100% MetOH.
4. Prepare the gel sandwich in the cassette for a Semi-dry transfer as follows: Negative electrode/Filter-Paper/Gel/Nitrocellulose-membrane/Filter-paper/Positive electrode. Remove any air bubbles between gel and membrane to ensure good results.
5. Place the cassette in the Semi-dry unit and run the blot at 25 V for 30 min. At the end, disassemble the blotting sandwich and store the membrane for the immunological assay. Check the transfer efficiency with Ponceau staining. Mark the membrane with a sign at the top right to preserve the right filter orientation.
6. Incubate with primary and secondary antibodies depending on the experiment (Figure 2).