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

Summary

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.

Abstract

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.

Introduction

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 transcription^3,4.

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 response^6,7,8. 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 approaches^5,8. MS relies on the detection of a difference in mass (Δm, 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 peptidoforms^17,18 (step 3). Altogether, the methods described here highlight the power of MS-based histone profiling in cancer epigenetics studies.

Protocol

Fresh frozen and OCT-frozen tissue biopsies were obtained under informed consent from patients undergoing surgery at the European Institute of Oncology (Milan), while FFPE biopsies were obtained from patients undergoing surgery at the Istituto Neurologico Carlo Besta (Milan). This study was approved by the Ethical Committee of the European Institute of Oncology (Study UID 2550) and of the Istituto Neurologico Carlo Besta (Study CET 39/24).

1. Histone extraction from patient-derived tissues (Figure 1A)

NOTE: To analyze histones from representative tumor cell populations and reduce inter-sample variability, it is advisable to use clinical biopsies with a tumor cellularity of at least 50% and devoid of necrotic areas and/or extensive immune infiltration.

1. Histone enrichment from fresh frozen tissues (estimated duration: 60 min)
   NOTE: All steps must be performed on ice.
   1. Thaw the tissue on ice and cut a piece of approximately 20-30 mg, usually corresponding to about 2 mm³ of tissue.
   2. Transfer the sample to a 1.5 mL tube and mince the tissue into small pieces using scissors (Table of Materials).
   3. Add 1 mL of Nuclei Isolation Buffer (Table 1) freshly supplemented with the protease inhibitors listed in the Table of Materials. Mix well afterwards.
   4. Transfer the sample to a Dounce homogenizer (Table of Materials).
      NOTE: In the presence of big tissue pieces, use a p1000 tip (upon cutting the tip with scissors) to better collect the sample.
   5. Homogenize the tissue by using first the LOOSE and then the TIGHT pestle (Table of Materials) until the tissue pieces have become invisible to the naked eye.
   6. Filter the homogenized sample through a 100 µm cell strainer (Table of Materials) to remove tissue debris.
   7. Pipette vigorously up and down with a p200 to dissolve the plasma membrane and transfer the sample to a new 1.5 mL tube.
   8. Pellet the cell nuclei by centrifugation at 2,300 x g for 15 min at 4 °C using a benchtop centrifuge (Table of Materials).
   9. Discard the supernatant and resuspend the nuclear pellets in 50-100 µL of Nuclei Isolation Buffer, supplemented with 0.1% SDS to dissolve the nuclear membrane (Table of Materials).
   10. Add 250 U of Benzonase nuclease to digest nucleic acids, mix well, and incubate for 2 min at 37°C (Table of Materials).
       NOTE: If the sample is still viscous, sonication can also be performed in a water bath (setting: 10 min 30s ON/30s OFF; power: high; temperature: 4 °C). After this step, samples can be stored at -20 °C.
2. Histone enrichment from OCT samples (estimated duration: 90 min)
   NOTE: This protocol has been optimized for four/five 10 µm-thick OCT sections corresponding to 20-60 mg of tissue. However, the number of tissue sections depends on the type of clinical biopsy and total tissue area. Considering the scarcity of clinical specimens, extraction from a lower amount of starting tissue can be performed, even though this might result in a decreased detection of histone modifications.
   1. Place four/five 10 µm-thick tissue sections, corresponding to approximately 20-60 mg of tissue, in a 1.5 mL tube.
   2. Wash the sections with 1 mL of ice-cold 70% EtOH (Table 1) for 2 min on a rotating wheel (Table of Materials) at 4 °C.
   3. Centrifuge at 16,000 x g for 2 min at 4 °C in a benchtop centrifuge.
   4. Repeat steps 1.2.2 and 1.2.3 a total of 3x.
   5. Rehydrate the samples in 1 mL of double-distilled water (ddH₂O) with rotation (Table of Materials) for 2 min at 4 °C.
   6. Centrifuge at 16,000 x g for 2 min at 4 °C in a benchtop centrifuge (Table of Materials).
   7. Repeat steps 1.2.5 and 1.2.6 twice with 1x PBS (Table of Materials).
   8. Proceed to step 1.1.2 of the enrichment protocol from fresh frozen samples.
3. Histone extraction from FFPE samples (estimated duration: 8 h)
   NOTE: The described protocol has been developed for four 10 µm-thick FFPE sections corresponding approximately to 20-60 mg of tissue. For most MS analyses of histone modifications, this amount is in excess and represents a safe starting point when the material is not limiting. However, less starting material can also be used, which is particularly relevant considering the often small amount of tissue available when analyzing clinical specimens. The use of less starting material, although feasible, typically reduces the quantification robustness for low-abundant histone modifications (e.g., H3K27ac, or multi-methylated H3K27/K36 peptides), and this effect can vary depending on the tissue type, as well as from sample to sample. Nevertheless, our lab has previously shown that MS-based histone PTM profiling can be successfully carried out from laser microdissected tissue areas of breast cancer FFPE samples corresponding to as low as 1,000 cells¹⁸.
   1. Place four 10 µm-thick FFPE sections, corresponding to approximately 20-60 mg of tissue, in a 1.5 mL tube.
   2. Add 1 mL of paraffin dissolving agent to the sample and vortex (Table of Materials) at maximum speed for 30 s, until paraffin is completely dissolved.
      CAUTION: Paraffin dissolving reagents are mutagenic, carcinogenic, and inflammable, so they must be used under a chemical hood.
      NOTE: Paraffin dissolution is achieved when paraffin pieces are no longer visible by the naked eye, and the paraffin dissolving agent has a whitish, homogeneous color.
   3. Centrifuge 16,000 x g for 3 min at RT in a benchtop centrifuge and discard the supernatant. To avoid undesirable sample aspiration during supernatant removal, vacuum pump/pipettes can be capped with a smaller tip (e.g., p200, p10 tips).
   4. Repeat Step 1.3.2 and 1.3.3 three additional times (4x in total) to ensure complete paraffin removal. Alternatively, weigh the completely de-paraffinized sample to have an indicative readout of the amount of tissue processed.
   5. Add 1 mL of 95% EtOH solution (Table 1) and vortex at maximum speed for 30 s, to ensure complete tissue resuspension.
   6. Centrifuge 16,000 x g for 3 min at RT in a benchtop centrifuge and discard the supernatant. Alternatively, weigh the dehydrated sample to have an indicative measure of the amount of dry tissue processed.
   7. Repeat step 1.3.5 and 1.3.6 with 70%, 50%, and 20% EtOH (Table 1) and ddH₂O to rehydrate the sample.
   8. Add 200 µL of Extraction buffer (Table 1).
      NOTE: If larger tissue pieces are present, cut them into smaller pieces with scissors. Depending on the amount of tissue, smaller/greater volumes of extraction buffer can be added to ensure proper sample lysis. For particularly small pellets resuspended in a volume of Extraction buffer lower than 100 µL, sonication can be performed using a sonication device (Table of Materials) (30 s ON/30 s OFF power: high, 10 cycles).
   9. Homogenize tissue in Extraction buffer by sonication using a digital sonifier (Table of Materials) with a 3 mm microtip (>15 cycles, 5s ON/2 min OFF, power: 15-30%).
      NOTE: Samples can be considered properly homogenized when no visible tissue particles are visible in the solution and the extraction buffer exhibits a uniform, non-turbid color. Keep samples at RT during sonication cycles. Ice may cause precipitation of SDS in the Extraction buffer, thus impairing tissue homogenization. Proper sonication is crucial to achieve complete tissue lysis and protein extraction. Place the microtip at a correct height in the sample tube (it must not touch the bottom of the tube, releasing plastic particles, and it must not be at the air-extraction buffer interface, creating foam and bubbles).
   10. After homogenization, incubate samples at 95 °C for 45 min in a thermomixer. Every 15 min, open the lid of the tubes to allow formaldehyde to evaporate.
   11. Incubate samples at 65 °C for 4 h in a thermomixer. Open the lid every 20-60 min to allow formaldehyde to evaporate.
   12. Centrifuge 16,000 x g for 1 min at RT in a benchtop centrifuge and transfer the supernatant into a new tube.
       NOTE: Samples can be stored at -20 °C at this point.

Table 1: Buffer Composition. Please click here to download this Table.

2. Histone resolution through SDS-PAGE and in-gel digestion

1. SDS-PAGE (Figure 1B; estimated duration: 2 h)
   1. To assess histone purity and estimate their quantity, load 1/10 of the histone extracts onto a 17% polyacrylamide gel (SDS-PAGE; Table 1) alongside known amounts of recombinant histones, as illustrated in Figure 1B.
      NOTE: Instead of loading 1/10 of histone extracts, 15-50 µg of proteins, as measured by assays that tolerate high detergent concentrations, such as the bicinchoninic acid (BCA) protein assay¹⁹ can be loaded. However, please note that histone enrichment over total proteins can vary significantly among fresh frozen, OCT frozen, and FFPE samples, and quantification of histone amount can only be based on SDS-PAGE. 1, 0.5, and 0.25 µg of recombinant histone H3.1 (Table of Materials) can be loaded as a reference to quantify the histone amount more precisely.
   2. Mix the samples with lithium dodecyl sulphate (LDS) loading buffer, or with equivalent loading buffers for SDS-PAGE (e.g., Laemmli buffer) with the addition of dithiothreitol (DTT; Table of Materials) at a final concentration of 10 mM.
   3. Denature samples at 95°C for 5 min in a thermomixer.
   4. After protein separation on the SDS-PAGE, stain the gel with Coomassie staining (Table of Materials).
   5. Estimate histone yield by visual or software-assisted²⁰ quantification of histone bands in clinical samples compared to recombinant histone H3 loaded at known amounts.
2. Histone in-gel digestion (Figure 2; estimated duration: 2 days)
   NOTE: ArgC-like digestion is employed for MS-analysis of histone PTMs because standard trypsin digestion generates excessively short peptides, due to the high frequency of lysine and arginine amino acids in their sequence, which hampers LC-MS detection of peptides and PTM identification. By chemically modifying lysines, the tryptic cleavage at these sites is prevented, resulting in cleavage at the C-terminus of arginines only, producing longer peptides (4-15 amino acids) that contain modified lysines within the sequence; overall, this protocol facilitates MS detection and measurement of modified histone peptides and has been thoroughly described in numerous publications^8,17,21.
   ​OPTIONAL: An internal standard (e.g., heavy labelled spike-in, synthetically labeled peptides²²) can be mixed with histone samples in a 1:1 ratio to achieve more accurate peptide quantification in MS (Figure 2B).
   1. Load 3-5 µg of histone sample on a 17% polyacrylamide gel, based on the estimated histone quantification performed in Step 2.1.5 (Figure 2B).
   2. After SDS-PAGE, visualize proteins by staining the gel with Coomassie staining.
      NOTE: To prevent contamination that may interfere with LC-MS/MS analysis, it is advisable to perform the following steps in a keratin-free environment, such as a dedicated hood, since keratin is a strong MS contaminant.
   3. Excise gel bands corresponding to the molecular weight of core histone proteins (gel band between 10 and 18 kDa, considering the following molecular weight for each core histones: H3 about 17 kDa, H2A-H2B about 14 kDa, H4 about 11 kDa) using a scalpel (Table of Materials) and cut bands in pieces of approximately 1 mm³ each.
   4. With the help of the scalpel, transfer the gel pieces to a 1.5 mL tube.
      NOTE: To completely cover gel pieces, the volumes indicated below can be increased, maintaining the reagent stoichiometry.
   5. Destain the gel pieces by adding a solution of 50% acetonitrile (ACN) in ddH₂O (Table 1). Place in a thermomixer set at 1,400 rpm for 10 min RT and discard the supernatant afterwards.
   6. Repeat Step 2.2.5 until the gel pieces are completely de-stained.
   7. Dehydrate the gel pieces by adding 100% ACN (Table of Materials). Incubate with mixing at 1,400 rpm for 10 min RT and discard the supernatant afterwards.
   8. Repeat step 2.2.7 until the gel pieces appear white and hard, thus completely dehydrated.
   9. Dry the gel pieces for 5 min at RT in a vacuum centrifuge (Table of Materials).
      NOTE: Histones are poor in cysteines (only two cysteines are found in histone H3 at positions 96 and 110 (UniprotKB ID P68431), and only one cysteine is found in histone H2B at position 154 (UniprotKB ID B4DR52)). Additionally, these cysteines are present within peptides not detectable upon ArgC-like digestion in bottom-up MS, so that chemical adducts of thiol groups²³ are not an artefact in MS analysis of histones, and no cysteine reduction and alkylation steps are performed in the described protocol.
   10. Add 15 µL of 7.7 M of propionic anhydride (PRO) solution (Table of Materials) and 26 µL of 1 M ammonium bicarbonate (AmBic) (Table of Materials) to each tube and incubate samples in a thermomixer set at 350 rpm for 10 min at 37 °C.
       NOTE: Since anhydrides dissolved in a reactive buffer tend to evaporate, do not prepare a master mix of PRO and AmBic, but add them individually to each sample, to maintain constant the proper concentration of PRO in each tube.
   11. Completely cover gel pieces by adding 1 M AmBic (recommended volume: 80 µL) and incubate samples with mixing at 1,400 rpm for 4 h at 37°C in a thermomixer. Remove all the liquid afterwards.
       ​NOTE: 1 M AmBic is added to maintain a basic pH, ideal for catalyzing the propionylation of unmodified and monomethylated lysines by PRO.
   12. Wash three times with ddH₂O, incubating samples in between at RT for 10 min while mixing in a thermomixer at 1,400 rpm. Discard liquid after each wash.
   13. Add 50% ACN and incubate in a thermomixer at 1,400 rpm for 15 min at RT. Discard the liquid afterwards.
   14. Completely dehydrate the gel pieces by adding 100% ACN and incubate at 1,400 rpm for 15 min at RT in a thermomixer. Discard the liquid afterwards.
   15. Repeat Step 2.2.14 until gel pieces appear white and dry.
   16. Dry gel pieces 5 min at RT in a vacuum centrifuge.
   17. Add to each sample 4 µL of trypsin solution (Table 1) and 20 µL of Digestion buffer (Table 1) and incubate 10 min on ice to allow trypsin absorption into gel pieces.
       NOTE: This incubation -carried out with concentrated trypsin solution and Digestion buffer (50 mM AmBic in ddH₂O, creating a basic pH ideal for trypsin cleavage) - allows the enzyme to be absorbed by the gel pieces prior to its activation at 37 °C.
   18. When concentrated trypsin is completely absorbed, cover the gel pieces thoroughly with additional Digestion buffer (indicative volume: 80 µL) and incubate at 37 °C overnight in a thermomixer.
   19. To extract digested peptides from the gel pieces, add 100 µL of 100% ACN and incubate at 1,400 rpm for 20 min at RT in a thermomixer.
   20. Collect the supernatant and transfer it to a new 1.5 mL tube.
   21. Add 80 µL of 100% ACN to gel pieces and incubate at 1,400 rpm for 10 min RT in a thermomixer.
   22. Collect supernatant and pool it with that collected in Step 2.2.20, then concentrate the peptides in a vacuum centrifuge to a volume between 1 and 5 µL.
       NOTE: The use of a vacuum centrifuge allows the evaporation of the organic solvent (ACN). However, it is recommended not to dry the sample completely at this stage, as this may negatively affect the following derivatization step. If the sample dries out, resuspend it in ddH₂O water and sonicate it in a water bath for 5 minutes.
   23. Add ddH₂O to each sample, to a final volume of 15 µL, then add 2 µL of 1 M triethylammonium bicarbonate (Table of Materials) and 3 µL of phenyl isocyanate (PIC) solution (Table 1) and incubate at 350 rpm for 90 min at 37 °C in a thermomixer.
       CAUTION: PIC is mutagenic and carcinogenic, so it is recommended to handle it under a chemical hood. Since PIC is highly reactive and volatile, it is recommended to avoid preparing a master mix of triethylammonium bicarbonate and PIC, but it is advisable to add each reagent individually to ensure the presence of the exact PIC concentration.
   24. Add to each tube 8 µL of 1 % trifluoroacetic acid (TFA; Table of Materials) to stop the derivatization with PIC.
   25. Dilute each sample with 100 µL of Buffer A (Table 1) and load them onto C18 stage tips (prepared as described in²⁴ or purchased from different providers).
   26. Spin the samples at 2,100 x g for 10 min at 4 °C in a swing rotor centrifuge to allow histone peptides to bind to the C18 resin.
       NOTE: Samples can be stored on Stage tips at 4 °C for several months.
   27. Add 30 µL of elution buffer (Table 1) to C18 Stage Tips and place tips into a new 1.5 mL tube. Spin samples at 1,000 x g for 10 min in a benchtop centrifuge at 4 °C to elute histone peptides.
   28. Concentrate samples to a volume between 1 and 3 µL using a vacuum centrifuge, then add 1% TFA to reach a final volume of 6 µL.
   29. Load 5 µL of peptide solution into a 96-well microplate for MS analysis (Table of Materials) and seal it with a sealing mat (Table of Materials).

3. HPLC-MS/MS and data analysis (Figure 3)

NOTE: The parameters hereby described are suited for an ESAY-nLC 1200 liquid chromatography system connected through an EASY Spray column to a Q Exactive Plus Orbitrap mass spectrometer (Table of Materials), but similar/equivalent instruments can also be used, with ad hoc parameters.

1. HPLC-MS/MS (estimated duration: 90 min per sample)
   1. Inject 3 µL out of the 5 µL histone peptides of step 2.2.29 (corresponding to approximately 0.25-0.8 µg, based on histone quantification by SDS-PAGE) onto a C18 nanocolumn (Table of Materials) at a constant flow of 500 nl/min in Buffer A.
   2. Apply a 55-min linear gradient of 10%-45% Buffer B at a flow rate of 250 nL/min to separate peptides.
   3. Acquire the MS/MS data in data-dependent mode (DDA) to automatically switch between MS1 and MS2 using the dedicated software packages (Table of Materials).
      NOTE: For acquisition and general settings, refer to Table 2.
2. RAW MS data analysis
   NOTE: In this protocol, the quantification of histone PTMs is carried out using EpiProfile, an open script (https://github.com/zfyuan/EpiProfile2.0_Family), user-friendly, and suitable for standard analyses as those described for H3 and H4 lysine methylations and acetylations. However, EpiProfile can be customized to carry out analyses of less standard modifications and upon different sample preparation protocols by more experienced users. Please note that EpiProfile requires a MATLAB license to be run²⁵.
   1. Analyze the acquired RAW data with EpiProfile 2.0 software (PRO-PIC version)²⁵ (Table of Materials) and quantify histone peptides by selecting the options histone_normal, when analysis is performed in label-free, and histone_SILAC, when using the SUPER-SILAC spike-in. Set ndebug = 0.
   2. For every histone peptide (e.g., H3_3-8), calculate the relative abundance (%RA) of each modified peptidoform (e.g., H3_3-8 unmod, me1, me2, me3) by dividing its extracted ion chromatogram (XIC) computed by EpiProfile by the sum of all the XICs computed for that peptide.
      NOTE: Optionally, XICs computed by EpiProfile can be validated by inspection of MS spectra and manual quantification of histone peptides using a dedicated spectra layout as described in^18,26.
   3. For each peptidoform, calculate the light-to-heavy (L/H) ratio by dividing the %RA of each native, light peptide by the %RA of the corresponding heavy peptide derived from the SUPER-SILAC mix, spiked into each sample.
   4. Transform the L/H ratios into a logarithmic scale. To highlight significant differences in peptidoform abundance across samples, statistical analysis can be performed, and the L/H ratios across all samples can be visually represented as heatmaps and scatter plots, as in Figure 4.

Table 2: List of the Acquisition settings and of the General setting employed for LC-MS/MS with the system used in this protocol. Please click here to download this Table.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Histolemon RS for Histology	Carlo Erba	454912	2.5 L
Acetonitrile (ACN)	Carlo Erba	412341	1L
Ammonium bicarbonate (AmBic)	Sigma Aldrich	A6141	500 g
Ammonium persulphate (APS)	WDR	1.012.010.500	500 g
Aprotinin protease inhibitor	Sigma Aldrich	A1153	5 mg
Benzonase nuclease	Merk	E1014	25 KU
Bisacrylamide 30% 37.5 M	AppliChem	APA36261000	1 L
Buffer A 0.1 % formic acid in ddH2O	Thermo Fisher Scientific	10229884	500 mL
Buffer B 0.1 % formic acid /80% acetonitrile in ddH2O	Thermo Fisher Scientific	15431423	500 mL
C18 resin	Merck	66883-U	47 mm diameter
Coomassie Instant Blue	Abcam	ab119211	1 L
Dithiotreitol (DTT)	WDR	441496P	25 g
Ethanol	Carlo Erba	414608	2.5 L
Leupeptin protease inhibitor	Sigma-Aldrich	L8511	5 mg
N,N,N′,N′-Tetramethyl ethylenediamine (TEMED)	Sigma Aldrich	1.107.320100	100 mL
NuPAGE LDS sample buffer	Invitrogen	NP0007	4x
PBS - Phosphate buffered saline	MicroGem	TL1006	500 mL
Phenil isocyanate (PIC)	Sigma Aldrich	185353	100 g
Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor	Sigma Aldrich	P7626	1 g
Propionic anhydride (PRO)	Sigma Aldrich	240311	50 g
Recombinant Histone H3.1 Human	New England Biolabs	M2503	1 µg/µL
Sequencing-grade trypsin	Promega	V5113	100 ug
Sodium butyrate (NaBut) histone deacetlyase inhibitor	Merck	B5887	5 g
Sodium dodecylsulphate (SDS)	Sigma Aldrich	L3771	100 g
Triethylammonium bicarbonate	Sigma Aldrich	T7408	100 mL
Trifluoroacetic acid (TFA)	Thermo Fisher Scientific	28904	1x10mL
Tris-HCl	Sigma Aldrich	T3253	100 g
Triton X-100 detergent	Sigma-Aldrich	X100RS	5 g
Kits/ Equipment/Software
96-well microplate	Corning	PCR-96-FS-C	Non-pyrogenic, Rnase/Dnase-free
Balance	Kern	19024
Benchtop centrifuge	Hareus Biofuge Lab	Pico, 23396
Bioruptor Sonication Device	Diagenode
Branson Digital Sonifier 250	Emerson
Cell strainer 100 µm	Falcon	352360
Concentrator plus	Eppendorf	5305000509
Dounce Homogenizer (e.g  Dounce, Wheaton...)	Dounce, Wheaton		1 mL Tissue Grinder
EASY-nLC 1200 Liquid Chromatography	Thermo Fisher Scientific
EASY-Spray HPLC C18 Column	Thermo Fisher Scientific	ES902	2 µm, 100  Å, 75 µm x 25 cm
EpiProfile2.0 script	https://github.com/zfyuan/EpiProfile2.0_Family
Fiji software	https://imagej.net/software/fiji/downloads
MATLAB	https://www.mathworks.com
MaxQuant search engine	https://maxquant.net/maxquant/
Pestel A (loose)	Dounce, Wheaton
Pestel B (tight)	Dounce, Wheaton
Q Exactive Plus Orbitrap LC-MS/MS system	Thermo Fisher Scientific
Rotating wheel	Thermo Fisher Scientific	11496548
Scalpel	Kiato	13010
Scissors	Thermo Fisher Scientific	15207266
Sealing Mat	Corning	AM-96-PCR-RD	Automation-compatible
Swing-out rotor centrifuge	Beckman coulter	Allegra X-15R
Thermomixer compact	Eppendorf	5350
Vortex	PBI	Vibromix
Xcalibur software	Thermo Fisher Scientific	OPTON-30965