Extraction of Histones from Clinical Specimens for Epigenetic Profiling by Mass Spectrometry

The scope of our research is to analyze epigenetic changes in cancer samples, discover new diagnostic and prognostic biomarkers, and identify potential target for therapy. A major challenge with clinical samples is the limited material and fixation-induced artifacts. However, with our optimized workflow and di sensitivity MS, we can quantify up to 50 histone PTMs from just 1000 cells.

We show that mass spectrometry can quantify histone PTM changes in clinical sample, offering insight into epigenetic mechanism and enabling biomarker discovery and drug target identification for precision epigenetic therapies. Our protocol addresses the need for our robust, quantitative and sensitive profiling on histone PTM in various type of clinical samples. Mass spectrometry is ideal for histone PTM analysis, offering unbiased detection, simultaneous profiling, and co-occurrence on the same peptide, thus overcoming the limitation of antibody-based methods.

To begin, thaw the fresh frozen tissue on ice and using a scalpel or blade, cut a piece of approximately 20 to 30 milligrams corresponding to around two cubic millimeters of tissue. Transfer the cut tissue sample to a 1.5 milliliter tube. Using scissors, mince the tissue into small pieces.

Add one milliliter of freshly prepared nuclei isolation buffer supplemented with the specified protease inhibitors and mince the tissue again. Transfer the sample to a down homogenizer. Using the loose pestle first, and then the tight pestle, homogenize the tissue until no visible pieces remain to the naked eye.

Filter the homogenized sample through a 100 micrometer cell strainer to eliminate tissue debris. Pipette the filtered sample vigorously up and down using a 200 microliter pipette to dissolve the plasma membrane. Transfer the sample to a new 1.5 milliliter tube.

Place the tube into a benchtop centrifuge and spin at 2, 300 G for 15 minutes at four degrees Celsius to pellet the nuclei. After discarding the supernatant, resuspend the nuclear pellet in 50 to 100 microliters of nuclei isolation buffer, supplemented with 0.1%SDS to dissolve the nuclear membrane. Then add 250 units of Benzonase nuclease to the resuspended sample to digest nucleic acids.

Mix well and incubate for two minutes at 37 degrees Celsius. For optimal cutting temperature frozen samples, wash the tissues with ethanol water and PBS before mincing and histone extraction. Add one milliliter of paraffin dissolving agent to the FFPE sample, taken in a 1.5 milliliter tube and vortex at maximum speed for 30 seconds until the paraffin is completely dissolved.

Centrifuge the sample at 16, 000 G for three minutes at room temperature and discard the supernatant carefully using a pipette fitted with a small tip avoiding the pellet. Now add one milliliter of 95%ethanol to the pellet and vortex at maximum speed for 30 seconds. Centrifuge the sample at 16, 000 G for three minutes at room temperature and discard the supernatant.

After rehydrating the sample, add 200 microliters of extraction buffer to the pellet. Homogenize the tissue in the extraction buffer by fornicating with a digital signifier using a three millimeter micro tip. After homogenization, incubate the sample in a thermo mixer and briefly open the tube lid to let the formaldehyde evaporate.

After centrifuging the sample, transfer the supernatant into a new labeled tube. Excise the gel bands corresponding to the molecular weight of core histone proteins located between 10 and 18 kilodaltons using a scalpel. After cutting the gel bands use the same scalpel to transfer the cut gel pieces into a 1.5 milliliter tube.

To destain the gel, add a solution of 50%of acetonitrile in double distilled water and incubate the tube in a thermo mixer at 1, 400 revolutions per minute for 10 minutes at room temperature. Dehydrate the gel pieces by adding 100%acetonitrile. Mix in the thermo mixer at 1, 400 revolutions per minute for 10 minutes at room temperature.

After discarding the supernatant, dry the gel pieces for five minutes at room temperature in a vacuum centrifuge. Next, add 15 microliters of 7.7 molar proponicanhydride solution, and 26 microliters of one molar ammonium bicarbonate to each tube and incubate. Then incubate the sample with 80 microliters of one molar ammonium bicarbonate.

Then wash the gel pieces three times with double distilled water and discard the liquid after each incubation. Next, add 50%acetone nitrile to the gel pieces and incubate at 1, 400 revolutions per minute for 15 minutes at room temperature. Then add 100%acetonitrile to completely dehydrate the gel pieces.

Incubate in the thermo mixer at 1, 400 revolutions per minute for 15 minutes at room temperature. After discarding the liquid, dry the gel pieces for five minutes at room temperature in a vacuum centrifuge. Add four microliters of trypsin solution and 20 microliters of digestion buffer to each tube containing gel pieces.

Incubate the tubes on ice for 10 minutes to allow trypsin absorption into the gel. Once the trypsin is fully absorbed, add 80 microliters of digestion buffer to completely cover the gel pieces and incubate the tubes overnight at 37 degrees Celsius in a thermo mixer. To extract the digested peptides, add 100 microliters of 100%aceto nitrile to each tube, and incubate at 1, 400 revolutions per minute for 20 minutes at room temperature in a thermo mixer.

Now, carefully collect the supernatant and transfer it into a new 1.5 milliliter tube. After repeating the aceto nitro extraction with the remaining gel, pool the supernatants. Concentrate the pooled peptides in a vacuum centrifuge until the volume is between one and five microliters.

Then add two microliters of one molar triethylammonium bicarbonate, and three microliters of phenyl isocyanate solution. Incubate the sample at 350 revolutions per minute for 90 minutes at 37 degrees Celsius in a thermo mixer. To stop the derivitization, add eight microliters of 1%trichloroacetic acid to each tube.

Finally, dilute each sample by adding 100 microliters of buffer A and load the diluted samples onto C18 stage tips for elution. Histone extraction was successfully achieved from fresh frozen, OCT frozen and FFPE samples using differential processing workflows specific to each tissue type. Despite lower enrichment and background protein presence, histone H3, H2A, H2B, and H4 were successfully extracted from FFPE samples as shown by bands in lane's FFPE1 and FFPE2, aligning with approximately one microgram of the H3.1 standard.

Nano liquid chromatography and MS analysis of FFPE derived histones showed successful separation of histone peptides over a 55 minute gradient with clear retention time peaks shown in the total ion chromatogram. Extracted ion chromatograms confirmed the detection and quantification of unmodified and methylated forms of H3 K four with separate elution profiles for each M by Z value. Enabling the calculation of percentage relative abundance for each modification.

Comparative analysis of histone modifications between normal and tumor samples revealed significant increases in certain types and a decrease in other types in tumor tissues.