Name: Extraction of Histones from Clinical Specimens for Epigenetic Profiling by Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2025-11-21T12:30:00.000+00:00
Duration: 10 min 54 s
Description: Learn histone extraction from clinical specimens for epigenetic profiling using mass spectrometry. This protocol quantifies up to 50 histone PTMs from just 1000 cells, enabling biomarker discovery and therapeutic target identification.

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5 minutes

We acknowledge surgeons and clinicians from the European Institute of Oncology and Istituto Neurologico Carlo Besta for providing FF, OCT frozen and FFPE tissue biopsies. We thank Roberta Noberini for the scientific and methodological discussion during manuscript preparation and our fundings: PRIN2022 (CUPG53D23001530006), AIRC (grant number IG-2023-28767), and Fondazione IEO-Monzino (FIEORDT-2023-BONALDI).

The authors declare no conflicts of interest.

The method described here provides a simple and robust workflow to efficiently isolate histones from clinical samples stored as fresh frozen, OCT frozen, and FFPE biopsies for the profiling of their modification landscapes by MS analysis. The subsequent sample preparation protocol via SDS-PAGE separation and PRO-PIC in-gel digestion with trypsin ensures effective removal of detergents, salts, and other MS contaminants33 and achieves accurate and robust quantification of histone lysine acetylations...

In the nucleus of eukaryotic cells, histones assemble with DNA to form the nucleosomes, the fundamental structural units of chromatin. Each nucleosome consists of approximately 146 bp of DNA wrapped around an octamer composed of two copies of core histones H2A and H2B, along with two dimers of histones H3 and H4. In addition, a linker histone H1 is also present and facilitates chromatin higher-order organization by binding both the nucleosome and the linker DNA stretch between nucleosomes1. Histones are decorated by a wide variety of post-translational modifications (PTMs) that are mainly present at their N-terminal tails. These modifications include methylation, various types of acylation (acetylation, propionylation, butyrylation, etc.), phosphorylation, SUMOylation, ubiquitylation, ADP-ribosylation, and crotonylation, among which lysine and arginine methylation and lysine acetylation are the most characterized2. The specific types, locations, and combinations of histone modifications create what is known as the histone code. This code consists of various post-translational modifications (PTMs) on the N-terminal tails of histones, which are added and removed by specific histone-modifying enzymes. These modifications are then recognized by effector proteins, leading to changes in chromatin structure, such as compaction or decondensation, ultimately influencing several biological processes, including DNA damage repair, replication, and particularly gene transcription3,4.
DNA methylation and histone PTMs are among the most studied epigenetic features in cancer3. Recent experimental evidence has shown alterations in the levels of various histone PTMs in several cancer types. Notably, the loss of H4K20me3 and H4K16ac has been reported as hallmarks of cancer, as their level was found to be decreased in essentially all tumors compared to the normal tissues4. Our group identified the loss of H3K14ac as a potential hallmark of cancer by quantifying its reduction in several cancers compared to normal counterparts using mass spectrometry (MS)-based approaches5. Moreover, numerous studies have highlighted the prognostic value of histone PTMs, showing that alterations in their global levels, primarily assessed by immunohistochemistry in patient biopsies, correlate with cancer progression, histological grading, and therapeutic response6,7,8. Hence, histone PTM analysis in cancer samples is important for the identification of novel diagnostic and prognostic biomarkers, as well as potential targets of the so-called epi-drugs8.
Very common methods for the analysis of histone PTMs in cell lines and clinical samples rely on antibody-based techniques, such as immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA)9. However, antibody-based methods suffer from various limitations, including cross-reactivity or, conversely, epitope masking10. Cross-reactivity occurs when antibodies bind to unintended targets with similar epitopes, leading to potential false positives, while epitope masking refers to the missed detection of a specific epitope (a histone PTM or variant, in this case) due to the presence of other modifications on the neighboring residues. Furthermore, the tissue amount required for histone modification analysis using antibodies is often high, since only a limited number of PTMs can be detected simultaneously, so several tissue slices must be processed for multiplexed analyses11. Mass spectrometry (MS) has emerged as the method of choice for profiling histone PTMs and variants, overcoming the limitations of antibody-based approaches5,8. MS relies on the detection of a difference in mass (&#916;m, delta mass) between the theoretical and experimentally measured masses of peptides and proteins. It is unbiased, comprehensive, and quantitative, as it allows the identification and quantification of PTMs and variants without requiring a priori knowledge of their site. This makes MS particularly valuable for the sensitive and accurate analysis of histone PTMs changes in cancer and other pathological conditions8.
Here we describe an optimized protocol for histone extraction from clinical samples, coupled with MS-based profiling of histone PTMs, thereafter referred to as epi-proteomics workflow. Patient biopsies are mainly stored as fresh frozen (FF) samples, optimal cutting temperature (OCT) frozen samples, and formalin-fixed paraffin-embedded (FFPE) samples12. While fresh biopsies are snap-frozen in liquid nitrogen or pre-cooled isopentane without the addition of any preservation medium13, OCT-frozen biopsies contain a water-soluble cryoprotective embedding medium, mainly composed of polyvinyl alcohol and polyethylene glycol, that facilitates tissue slicing14. FFPE samples, on the other hand, fixed in formalin and embedded in paraffin, represent the most common storage method for clinical specimens, as they can be stored at room temperature for several years, avoiding costly maintenance at low or ultra-low temperatures15. Being free of preservation additives, snap-frozen biopsies can be directly processed for histone extraction, while OCT-frozen and FFPE biopsies require initial steps of embedding medium/fixative removal. FFPE samples also need extensive protein decrosslinking and antigen retrieval.
This protocol outlines detailed methods for extracting histones from fresh-frozen, OCT-embedded, and FFPE clinical samples, ensuring sufficient quantity and quality for downstream MS analysis by taking into consideration the specific challenges associated with each type of sample preservation (step 1). Histones purified from clinical samples are separated through protein gel electrophoresis in denaturing conditions (SDS-PAGE), followed by a derivatization step, and enzymatically digested to generate peptides of a suitable length (4-15 amino acids) for bottom-up mass spectrometry analysis16 (step 2). More specifically, histones are digested with an ArgC-like strategy, whereby trypsin, that usually cuts at the C-terminus of unmodified lysines (K) and arginines (R), is induced to cut only at the C-terminus of arginines, due to a previous chemical acylation of unmodified and monomethylated lysines with propionic anhydride (PRO). Next, Arg-C-like digested histone peptides are acylated at their N-terminal tails by the addition of phenyl isocyanate (PIC). This further derivatization increases peptide hydrophobicity and allows better separation of short histone peptides by reverse-phase high-performance liquid chromatography (RP-HPLC), improving the identification and quantification of isobaric peptidoforms17,18 (step 3). Altogether, the methods described here highlight the power of MS-based histone profiling in cancer epigenetics studies.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Histolemon RS for Histology</td><td>Carlo Erba</td><td>454912</td><td>2.5 L</td></tr><tr><td>Acetonitrile (ACN)</td><td>Carlo Erba</td><td>412341</td><td>1L</td></tr><tr><td>Ammonium bicarbonate (AmBic)</td><td>Sigma Aldrich</td><td>A6141</td><td>500 g</td></tr><tr><td>Ammonium persulphate (APS)</td><td>WDR</td><td>1.012.010.500</td><td>500 g</td></tr><tr><td>Aprotinin protease inhibitor</td><td>Sigma Aldrich&amp;nbsp;</td><td>A1153</td><td>5 mg</td></tr><tr><td>Benzonase nuclease</td><td>Merk</td><td>E1014</td><td>25 KU</td></tr><tr><td>Bisacrylamide 30% 37.5 M</td><td>AppliChem</td><td>APA36261000</td><td>1 L</td></tr><tr><td>Buffer A 0.1 % formic acid in ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>10229884</td><td>500 mL&amp;nbsp;</td></tr><tr><td>Buffer B 0.1 % formic acid /80% acetonitrile in ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>15431423</td><td>500 mL</td></tr><tr><td>C18 resin</td><td>Merck</td><td>66883-U</td><td>47 mm diameter</td></tr><tr><td>Coomassie Instant Blue</td><td>Abcam</td><td>ab119211</td><td>1 L</td></tr><tr><td>Dithiotreitol (DTT)&amp;nbsp;</td><td>WDR</td><td>441496P</td><td>25 g</td></tr><tr><td>Ethanol</td><td>Carlo Erba</td><td>414608</td><td>2.5 L</td></tr><tr><td>Leupeptin&amp;nbsp;protease inhibitor</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L8511</td><td>5 mg</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl ethylenediamine (TEMED)</td><td>Sigma Aldrich</td><td>1.107.320100</td><td>100 mL</td></tr><tr><td>NuPAGE LDS sample buffer</td><td>Invitrogen</td><td>NP0007</td><td>4x</td></tr><tr><td>PBS - Phosphate buffered saline&amp;nbsp;</td><td>MicroGem</td><td>TL1006</td><td>500 mL</td></tr><tr><td>Phenil isocyanate (PIC)</td><td>Sigma Aldrich</td><td>185353</td><td>100 g</td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor</td><td>Sigma Aldrich&amp;nbsp;</td><td>P7626</td><td>1 g</td></tr><tr><td>Propionic anhydride (PRO)</td><td>Sigma Aldrich</td><td>240311</td><td>50 g</td></tr><tr><td>Recombinant Histone H3.1 Human</td><td>New England Biolabs</td><td>M2503</td><td>1 &amp;micro;g/&amp;micro;L</td></tr><tr><td>Sequencing-grade trypsin</td><td>Promega</td><td>V5113</td><td>100 ug</td></tr><tr><td>Sodium butyrate (NaBut) histone deacetlyase inhibitor</td><td>Merck</td><td>B5887</td><td>5 g</td></tr><tr><td>Sodium dodecylsulphate (SDS)</td><td>Sigma Aldrich</td><td>L3771</td><td>100 g</td></tr><tr><td>Triethylammonium bicarbonate</td><td>Sigma Aldrich</td><td>T7408</td><td>100 mL</td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Thermo Fisher Scientific</td><td>28904</td><td>1x10mL</td></tr><tr><td>Tris-HCl</td><td>Sigma Aldrich</td><td>T3253</td><td>100 g</td></tr><tr><td>Triton X-100 detergent</td><td>Sigma-Aldrich&amp;nbsp;</td><td>X100RS</td><td>5 g</td></tr><tr><td>&lt;strong&gt;Kits/ Equipment/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well microplate</td><td>Corning</td><td>PCR-96-FS-C</td><td>Non-pyrogenic, Rnase/Dnase-free</td></tr><tr><td>Balance</td><td>Kern</td><td>19024</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Hareus Biofuge Lab</td><td>Pico, 23396</td><td></td></tr><tr><td>Bioruptor Sonication Device</td><td>Diagenode</td><td></td><td></td></tr><tr><td>Branson Digital Sonifier 250</td><td>Emerson</td><td></td><td></td></tr><tr><td>Cell strainer 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Concentrator plus</td><td>Eppendorf</td><td>5305000509</td><td></td></tr><tr><td>Dounce Homogenizer (e.g&amp;nbsp; Dounce, Wheaton...)</td><td>Dounce, Wheaton</td><td></td><td>1 mL Tissue Grinder</td></tr><tr><td>EASY-nLC 1200 Liquid Chromatography</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>EASY-Spray HPLC C18 Column</td><td>Thermo Fisher Scientific</td><td>ES902</td><td>2 &amp;micro;m, 100&amp;nbsp; &amp;Aring;, 75 &amp;micro;m x 25 cm</td></tr><tr><td>EpiProfile2.0 script</td><td>https://github.com/zfyuan/EpiProfile2.0_Family</td><td></td><td></td></tr><tr><td>Fiji software</td><td>https://imagej.net/software/fiji/downloads</td><td></td><td></td></tr><tr><td>MATLAB</td><td>https://www.mathworks.com</td><td></td><td></td></tr><tr><td>MaxQuant search engine</td><td>https://maxquant.net/maxquant/</td><td></td><td></td></tr><tr><td>Pestel A (loose)</td><td>Dounce, Wheaton</td><td></td><td></td></tr><tr><td>Pestel B (tight)</td><td>Dounce, Wheaton</td><td></td><td></td></tr><tr><td>Q Exactive Plus Orbitrap LC-MS/MS system</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Rotating wheel</td><td>Thermo Fisher Scientific</td><td>11496548</td><td></td></tr><tr><td>Scalpel</td><td>Kiato</td><td>13010</td><td></td></tr><tr><td>Scissors</td><td>Thermo Fisher Scientific</td><td>15207266</td><td></td></tr><tr><td>Sealing Mat</td><td>Corning</td><td>AM-96-PCR-RD</td><td>Automation-compatible</td></tr><tr><td>Swing-out rotor centrifuge</td><td>Beckman coulter</td><td>Allegra X-15R</td><td></td></tr><tr><td>Thermomixer compact</td><td>Eppendorf</td><td>5350</td><td></td></tr><tr><td>Vortex</td><td>PBI</td><td>Vibromix</td><td></td></tr><tr><td>Xcalibur software</td><td>Thermo Fisher Scientific</td><td>OPTON-30965</td><td></td></tr></tbody></table>

extraction of histones from clinical specimens for epigenetic profiling by mass spectrometry

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

Extensive experimental evidence supports the role of epigenetic mechanisms in cancer onset, progression, and recurrence. Among these, histone variants and their post-translational modifications (PTMs) regulate chromatin organization and dynamics, and genome accessibility, thereby influencing key DNA-based processes, such as transcription, replication, and DNA damage repair through the so-called histone code. Dysregulated histone PTM patterns are frequently observed in cancers, with some markers that have been shown to serve as diagnostic and prognostic biomarkers. Thus, novel methodologies enabling unbiased and comprehensive histone PTM profiling in clinical samples are highly valuable for characterizing the epigenetic landscapes of tumors. Here, we present a protocol for the efficient extraction of histones from clinical samples, including snap frozen, optimal-cutting temperature (OCT) frozen, and formalin-fixed paraffin-embedded (FFPE) biopsies. This approach yields histone proteins of sufficient quantity and quality for the subsequent bottom-up analysis by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This workflow enables the robust detection and quantification of key histone modifications, particularly lysine acetylations and methylations, across primary tumor samples, providing an additional molecular layer for cancer characterization.

We have performed histone extraction from fresh frozen and OCT frozen breast cancer samples and from FFPE meningioma samples following the above-mentioned protocol. As illustrated in Figure 1A, for frozen specimens, we removed OCT, when present, and we homogenized samples in Nuclei isolation buffer containing a mild detergent (0.1% Triton step 1.1), allowing nuclei isolation. Next, we extracted histones with the addition of 0.1% SDS; instead, for FFPE specimens, we de-paraffinized, rehydrate...

This protocol enables efficient histone extraction from various clinical samples for subsequent post-translational modification analysis by LC-MS/MS. By facilitating robust detection of key histone modifications, it serves as a valuable tool for comprehensive epigenetic profiling in large clinical cohorts.

Extensive experimental evidence supports the role of epigenetic mechanisms in cancer onset, progression, and recurrence. Among these, histone variants and ...

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.

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Learn histone extraction from clinical specimens for epigenetic profiling using mass spectrometry. This protocol quantifies up to 50 histone PTMs from just 1000 cells, enabling biomarker discovery and therapeutic target identification.

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

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