Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Summary

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Abstract

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Introduction

Since the late 19^th century, cell culture systems have been used as a model to study the growth and development of cells outside of the human body^1,2. Their use has also been extended to study how tissues and organs function in both healthy and diseased contexts^1,3. Suspension cells (e.g., blood cells) grow in Petri dishes or flasks seamlessly and interchangeably as they do not assemble in three-dimensional (3D) structures in vivo. Cells derived from solid organs can grow in either two-dimensional (2D) or 3D culture systems. In 2D culture, cells are grown in a monolayer that adheres to a flat surface^2,4. 2D cell culture systems are characterized by exponential growth and a fast doubling time, typically 24 h to a few days⁵. Cells in 3D systems grow to form intricate cell-cell interactions modeling tissue-like conglomerates more closely, and they are characterized by their ability to reach a dynamic equilibrium where their doubling time can reach 1 month or longer⁵.

Presented in this paper is an innovative methodology to grow 3D spheroids in rotating cell culture systems that simulate reduced gravity⁶. This is a simplified derivative of a cell culture system introduced by NASA in the 1990s⁷. This approach minimizes shearing forces, which occur in existing methods like spinning flasks, and increases spheroid reproducibility⁶. In addition, the rotating bioreactor increases active nutrient diffusion, minimizing necrotic formation that occurs in systems like hanging drop cell culture where media exchange is impractical⁶. This way, cells grow mostly undisturbed, allowing for the formation of structural and physiological characteristics associated with cells growing in tissue. C3A hepatocytes (HepG2/C3A) cultured in this manner not only had ultrastructural organelles, but also produced amounts of ATP, adenylate kinase, urea, and cholesterol comparable to levels observed in vivo^1,2. In addition, cells grown in 2D vs. 3D cell culture systems exhibit different gene expression patterns⁸. Gene expression analysis of C3A hepatocytes grown as 3D spheroids showed that these cells expressed a wide range of liver-specific proteins, as well as genes involved in key pathways that regulate liver function⁸. Prior publications demonstrated the differences between proteomes of exponentially growing cells in 2D culture vs. cells at dynamic equilibrium in 3D spheroid cultures⁵. These differences include cellular metabolism, which in turn affects the structure, function, and physiology of the cell⁵. The proteome of cells grown in 2D culture was more enriched in proteins involved in cell replication, while the proteome of 3D spheroids was more enriched in liver functionality⁵.

The slower replication rate of cells grown as 3D spheroids more accurately models specific phenomena associated with chromatin state and modifications (e.g. histone clipping⁹). Histone clipping is an irreversible histone post-translational modification (PTM) that causes proteolytic cleavage of part of the histone N-terminal tail. While its biological function is still under discussion^10,11,12,13, it is clear that its presence in primary cells and liver tissue is modeled by HepG2/C3A cells grown as spheroids, but not as flat cells⁹. This is critical, as chromatin state and modifications regulate DNA readout mostly by modulating accessibility to genes and thus their expression¹⁴. Histone PTMs either influence chromatin state directly by affecting the net charge of the nucleosomes where histones are assembled, or indirectly by recruiting chromatin writers, readers, and erasers¹⁴. Hundreds of histone PTMs have been identified to date¹⁵, reinforcing the hypothesis that chromatin hosts a "histone code" used by the cell to interpret DNA¹⁶. However, the identification of a myriad of PTM combinations¹⁵, and the discovery that combinations of histone PTMs frequently have different biological functions from PTMs present in isolation (e.g. Fischle, et al.¹⁷), highlights that more work is required to decrypt the "histone code".

Currently, histone PTM analysis is either based on techniques utilizing antibodies (e.g., western blots, immunofluorescence, or chromatin immunoprecipitation followed by sequencing [ChIP-seq]) or mass spectrometry (MS). Antibody-based techniques have high sensitivity and can provide detailed information about the genome-wide localization of histone marks but are frequently limited in studying rare PTMs or PTMs present in combinations^18,19,20. MS is more suitable for high-throughput and unbiased identification and quantification of single and co-existing protein modifications, in particular histone proteins^18,19,20. For these reasons, this and several other laboratories have optimized the MS pipeline for the analysis of histone peptides (bottom-up MS), intact histone tails (middle-down MS), and full-length histone proteins (top-down MS)^21,22,23.

Detailed below is a workflow for growing HepG2/C3A spheroids and preparing them for histone peptide analysis (bottom-up MS) via nano-liquid chromatography, coupled online with tandem mass spectrometry (nLC-MS/MS). A 2D cell culture was grown and the cells were harvested and transferred to a bioreactor where they would start to form spheroids (Figure 1). After 18 days in culture, spheroids were treated with sodium butyrate or sodium succinate to increase the relative abundances of histone acetylation and succinylation. Notably, 3D cultures can be treated with genotoxic compounds just as well as their flat culture equivalents; in fact, recent publications highlight that the toxicology response of cells in 3D culture is more similar to primary tissues than those in 2D flat culture^24,25. Cells were then collected at specified time points and nuclear isolation was performed. Then, histones were extracted and derivatized with propionic anhydride before and after trypsin digestion according to a protocol first developed by Garcia et al.²⁶. This procedure generates peptides of an appropriate size for online separation with reversed-phase chromatography (C₁₈) and detection with MS. Finally, histone peptides were identified and quantified, and the generated data was represented in multiple ways for a more complete biological interpretation.

Protocol

1. Preparation of buffers and reagents

1. Cell growth media (for HepG2/C3A cells): Add fetal bovine serum (FBS) (10% v/v), non-essential amino acids (1% v/v), L-glutamine supplement (1% v/v) and penicillin/streptomycin (0.5% v/v) to Dulbecco's Modified Eagle's Medium (DMEM, containing 4.5 g/L glucose). Growth media is stored at 4 °C for a maximum of 2 weeks.
2. 200 mM sodium butyrate (NaBut) solution: To prepare 10 mL, resuspend 220.18 mg of NaBut in 10 mL of ddH₂O. Store 1 mL aliquots at -20 °C. Before cell treatment, filter the solution using a 0.45 mm syringe filter and add 1 mL of the filtered solution to 9 mL of cell growth media for a working concentration of 20 mM.
3. 100 mM sodium succinate (NaSuc) solution: To prepare 10 mL, resuspend 162.05 mg of NaSuc in 10 mL of ddH₂O. Store 1 mL aliquots at -20 °C. Before cell treatment, filter the solution using a 0.45 mm syringe filter and add 1 mL of the filtered solution to 9 mL of cell growth media for a working concentration of 10 mM.
4. Cold 0.2 M H₂SO₄: To prepare 1 L, add 10 mL of concentrated H₂SO₄ to 990 mL of HPLC grade water. Store at 4 °C.
5. Cold acetone + 0.1% hydrochloric acid (HCl): Add concentrated HCl (0.1% v/v) to acetone. Store at 4 °C.
6. 100 mM NH₄HCO₃ solution, pH 8.0: To prepare 1 L, resuspend 7.91 g of NH₄HCO₃ in 1 L of HPLC grade water. Store 50 mL aliquots at -20 °C.
7. 0.1% Trifluoroacetic acid (TFA) solution: Add concentrated TFA (0.1% v/v) to HPLC grade water. Store at 4 °C.
8. 60% acetonitrile/0.1% TFA solution: Add HPLC grade acetonitrile (60% v/v) to HPLC grade water. Then, add concentrated TFA (0.1% v/v) to this solution. Store at 4 °C.
9. 2% HPLC-grade acetonitrile + 0.1% formic acid: Add HPLC-grade acetonitrile (2% v/v) to HPLC-grade water. Then, add concentrated formic acid (0.1% v/v) to this solution.
10. 80% HPLC-grade acetonitrile + 0.1% formic acid: Add HPLC-grade acetonitrile (80% v/v) to HPLC-grade water. Then, add concentrated formic acid (0.1% v/v) to this solution.

2. Preparation of the 3D culture system

NOTE: Different cells, primary or immortalized, have different culture properties, so the formation of spheroids may differ among cell types. This protocol has been established for HepG2/C3A spheroid formation using bioreactors and an innovative 3D cell culture system.

1. Using standard growth media, grow cells as a monolayer until they are 80% confluent.
2. Wash cells with HBSS (5 mL for a 75 cm² flask) and incubate cells with 5 mL of 0.05% trypsin-EDTA diluted in HBSS (1:2 dilution) for 5 min at 37 ^oC with 5% CO₂.
3. Check cell detachment under a microscope and neutralize trypsin by adding 3 mL of fetal bovine serum (FBS) or growth media (containing 5%-10% FBS).
4. Count the number of cells and dilute the cell suspension to obtain 1 x 10⁶ cells in a maximum volume of 1.5 mL.
5. Equilibrate an ultra-low attachment 24-well round bottom plate (containing multiple micro-wells per well) by washing the wells with 0.5 mL of growth media. Centrifuge the plate for 5 min at 3,000 x g to remove air bubbles from the well's surface.
6. Transfer the cell suspension into the plate and centrifuge for 3 min at 120 x g.
7. Incubate the plate for 24 h at 37 ^oC with 5% CO₂ for spheroid formation. Meanwhile, equilibrate the bioreactor by filling the humidity chamber with 25 mL of sterile water and the cell chamber with 9 mL of growth media.
8. Incubate the bioreactor, rotating in the clinostat incubator, for 24 h at 37 ^oC with 5% CO₂.

3. Growth of spheroids in bioreactors

NOTE: To preserve the structure of spheroids, wide bore tips are used for handling the 3D structures.

1. Detach spheroids from the ultra-low attachment plate by gently pipetting up and down with 1 mL wide bore tips and transfer to a tissue culture-treated dish.
2. Wash the plate with 0.5 mL of pre-warmed growth media and transfer to the same dish.
3. Evaluate the quality of spheroids by microscopy and select sufficiently formed spheroids. Good quality spheroids have uniform size, compactness, and roundness.
4. Transfer spheroids into equilibrated bioreactors filled with 5 mL of fresh growth media. After transferring the spheroids, completely fill the bioreactor with fresh growth media.
5. Place bioreactor in the clinostat incubator and adjust the rotation speed to 10-11 rpm.
6. Exchange growth media every 2-3 days by removing 10 mL of old media and replacing it with 10 mL of fresh media.
7. Adjust rotation speed according to spheroid growth, increasing as spheroids grow in size and number.
8. After 18 days in culture, spheroids are ready for treatment and/or collection

4. Spheroid treatment and collection

NOTE: In this protocol, HepG2/C3A spheroids are treated with sodium butyrate (NaBut) and sodium succinate (NaSuc) to evaluate the levels of histone marks containing acetylation and succinylation, respectively.

1. Prepare growth media with the appropriate working concentration of compound (e.g., 20 mM of NaBut or 10 mM NaSuc). Exchange the media in the bioreactor with the treated media.
   NOTE: To establish a control condition, either collect enough spheroids for experiments before adding the treatment or designate a bioreactor for untreated spheroids.
2. Collect spheroids for proteomic analysis following sufficient treatment time (e.g., 48 h to 1 week for NaSuc treatment or 48-72 h for NaBut treatment).
   NOTE: For histone extraction using this protocol, six to eight spheroids containing approximately 1 x 10⁶ cells were collected.
   1. Remove 3-5 mL of media from the bioreactor through the top port.
   2. Open the front port and use a 1 mL wide bore tip to remove spheroids and place them into microcentrifuge tubes.
   3. Centrifuge the spheroids at 100 x g for 5 min and discard media.
      NOTE: The media in the bioreactor can be changed and the bioreactor can be returned to the incubator for additional treatment time or recovery.
   4. Wash spheroids with 200 µL of HBSS to remove the FBS. Centrifuge at 100 x g for 5 min and remove the supernatant.
      ​NOTE: Spheroids can be stored at -80 ^oC until processing.

5. Histone extraction

NOTE: The many basic amino acid residues present in histones allow them to closely interact with DNA, which has a phosphoric acid backbone. Because histones are some of the most basic proteins in the nucleus, when they are extracted with ice-cold sulfuric acid (0.2 M H₂SO₄) there is minimal contamination. The non-histone proteins will precipitate in strong acid. Highly concentrated trichloroacetic acid (TCA) diluted to a final concentration of 33% is used afterward to precipitate the histones from the sulfuric acid. Keep all samples, tubes, and reagents on ice for the entire histone extraction.

1. Add five volumes (~100 µL) of cold 0.2 M H₂SO₄ to the cell pellet (~10-20 µL), and pipette up and down to disrupt the pellet and release histones.
2. Incubate tubes for up to 4 h at constant rotation or shaking at 4 ^oC.
   NOTE: For resuspended samples that have a volume greater than 500 µL, a 2 h incubation is sufficient to extract histones (longer incubation may result in extraction of other basic nuclear proteins). For resuspended samples with a volume less than 200 µL, a 4 h incubation is required for a better yield.
3. Centrifuge at 3,400 x g for 5 min at 4 °C. Collect supernatant in a new tube and discard the pellet later.
4. Add cold concentrated TCA such that it makes up a final 25%-33% v/v (e.g., 40-60 µL of cold TCA: 120 µL of supernatant) and mix by inverting the tube a few times.
5. Incubate tubes for at least 1 h at constant rotation or shaking at 4 ^oC.
   NOTE: For smaller starting pellet sizes, an overnight incubation is recommended.
6. Centrifuge at 3,400 x g for 5 min at 4 ^oC. Discard supernatant by pipetting. Aspirate the supernatant carefully; do not touch the sides of the tube or the pellet.
   ​NOTE: Histones deposit at both the sides and the bottom of the tube. The white insoluble pellet formed at the very bottom of the tube contains mostly non-histone proteins and other biomolecules.
7. Wash the tube (walls and pellet) with cold acetone + 0.1% HCl using a glass Pasteur pipette (~500 µL/tube).
8. Centrifuge at 3,400 x g for 5 min at 4 ^oC. Discard supernatant by flipping the tube.
9. Wash the tube (walls and pellet) with 100% cold acetone using a glass Pasteur pipette (~500 µL/tube). Centrifuge at 3,400 x g for 5 min at 4 ^oC.
10. Discard supernatant by flipping the tube and pipette out the remaining acetone. Open the lid and air-dry the sample on the bench for ~20 min.
11. Proceed to propionylation or store samples in -80 ^oC until use.

6. First round of derivatization

NOTE: The use of trypsin to digest histone proteins leads to excessively small peptides that are difficult to identify using traditional proteomics setups. For this reason, propionic anhydride is used to chemically derivatize the ɛ-amino groups of unmodified and monomethyl lysine residues. This restricts trypsin proteolysis to C-terminal arginine residues. For samples in 96-well plates, the use of multi-channel pipettes and reservoirs for reagent pick up is recommended (Figure 2A). Derivatization is also performed after digestion to label the free N-termini of the peptides increasing peptide hydrophobicity and thus reversed-phase chromatographic retention.

1. Resuspend samples in 20 µL of 15%-20% acetonitrile in 100 mM ammonium bicarbonate (pH 8.0). Vortex for 15 s, and then spin down at 1,000 x g for 30 s.
2. If there are eight or more samples, transfer each resuspended sample into a 96-well plate.
   NOTE: If the samples have not been transferred to a 96-well plate, a single channel pipette can be used in steps 6.3-6.7, 7.1-7.5, and 8.1-8.5.
3. Under the hood, add 2 µL of propionic anhydride using a multichannel pipette. Mix by pipetting up and down 5x.
4. Quickly add 10 µL of ammonium hydroxide using a multichannel pipette. Mix by pipetting up and down 5x.
   NOTE: Propionic acid is a product of the reaction between propionic anhydride and free amines from peptides and can decrease sample pH. pH 8 can be re-established by adding ammonium hydroxide to the sample at a ratio of 1:5 (v/v).
5. Ensure the pH is 8 using pH test paper. If pH < 8, adjust by adding 1 µL of ammonium hydroxide. If pH > 8, adjust by adding 1 μL formic or acetic acid. When pH > 10, labeling of other amino acid residues with higher pKa is possible.
6. Incubate at room temperature for 10 min.
7. Repeat steps 6.3-6.6. A double round of histone propionylation ensures nearly complete reaction efficiency.
8. Dry plate in speed vacuum until all wells are completely dried (~9 h).
9. Proceed to trypsin digestion or store samples at -80 ^oC until use.

7. Histone digestion

NOTE: Histones are digested into peptides using trypsin, which cuts at the carboxyl side of arginine and lysine residues. However, since propionylation modifies lysine residues, only arginine residues are cleaved (Figure 2B).

1. Prepare trypsin solution (25 ng/µL in 50 mM NH₄HCO₃, pH 8.0). Add 20 µL of trypsin (500 ng) to each sample.
   1. To prepare 50 mM NH₄HCO₃ solution, dilute 100 mM NH₄HCO₃ solution 1:1 v/v with HPLC grade water.
2. Ensure pH is 8 using pH test paper. If pH < 8, adjust by adding 1 µL of ammonium hydroxide. If pH > 8, adjust by adding 1 μL of formic or acetic acid.
3. Digest at room temperature overnight or incubate at 37 ^oC for 6-8 h.
4. If feasible, check the pH after ~3 h of digestion, as it may have decreased. If pH < 8, adjust by adding 1 µL of ammonium hydroxide.
5. Add an additional 5 µL of 50 ng/µL trypsin solution (250 ng) and continue digestion.
6. Proceed to the second round of propionylation or store samples at -80 ^oC until use.

8. Derivatization of peptide N-termini

NOTE: The propionylation of histone peptides at their N-terminus improves retention of the shortest peptides by reversed-phase liquid chromatography (e.g., amino acids 3-8 of histone H3), as the propionyl group increases peptide hydrophobicity.

1. Under the hood, add 2 µL of propionic anhydride using the multichannel pipette. Mix by pipetting up and down 5x.
2. Quickly add 10 µL of ammonium hydroxide using the multichannel pipette. Mix by pipetting up and down 5x.
   NOTE: Propionic acid is a product of the reaction between propionic anhydride and free amines from peptides, and can decrease sample pH. pH 8 can be re-established by adding ammonium hydroxide to the sample at a ratio of 1:5 (v/v).
3. Ensure the pH is 8 using pH test paper. If pH < 8, adjust by adding 1 µL of ammonium hydroxide. If pH > 8, adjust by adding 1 μL formic or acetic acid. When pH > 10, labeling of other amino acid residues with higher pKa is possible.
4. Incubate at room temperature for 10 min.
5. Repeat steps 8.1-8.4. A double round of histone propionylation ensures nearly complete reaction efficiency.
6. Dry plate in speed vacuum until all wells are completely dried (~9 h).
7. Proceed to the desalting step or store samples at -80 ^oC until use.

9. Desalting and sample cleanup

NOTE: Salts that are present in the sample interfere with mass spectrometry analysis. Salts are also ionized during electrospray and can suppress signals from peptides. Salts can form ionic adducts on peptides, which cause the adducted peptide to have a different mass. This reduces the peptide's signal intensity and prevents proper identification and quantification. The setup for desalting is illustrated in Figure 2C.

1. Start mixing the HLB resin (50 mg/mL in 100% acetonitrile) on a magnetic stir plate.
2. Make sure there is a 96-well collection plate placed underneath the 96-well polypropylene filter plate to collect the flow-through.
3. Add 70 µL of HLB suspension per well to the filter plate. Gently turn on the vacuum to prevent splashing. Discard the flow-through.
4. Wash the resin with 100 µL of 0.1% TFA. Gently turn on the vacuum to prevent splashing. Discard the flow-through.
5. Resuspend each sample in 100 µL of 0.1% TFA. Check the pH; it should be ~2-3.
6. Load each sample into each well. Turn on the vacuum gently to prevent splashing. Discard the flow-through.
7. Wash with 100 µL 0.1% TFA. Turn on the vacuum gently to prevent splashing. Discard the flow-through.
8. Replace the collection plate with a new 96-well collection plate.
9. Add 60 µL of 60% acetonitrile/0.1% TFA per well. Turn on the vacuum gently to prevent splashing. Collect the flow-through and dry in a speed vacuum.
10. Proceed to LC-MS/MS or store samples at -80 ^oC until use.

10. Histone peptide analysis  via liquid chromatography coupled with mass spectrometry

1. Prepare the mobile phases to run on the high-performance liquid chromatography (HPLC). Mobile Phase A (MPA): 2% HPLC-grade acetonitrile + 0.1% formic acid. Mobile Phase B (MPB): 80% HPLC-grade acetonitrile + 0.1% formic acid.
2. Program the HPLC method as follows: (1) 4%-34% MPB over 30 min; (2) 34%-90% MPB over 5 min; and (3) isocratic 90% MPB for 5 min. Use the following recommended column properties: C₁₈ 3 µm packing material, 75 µm internal diameter, 20-25 cm length. Set the flow rate to 250-300 nL/min for nano-columns with a 75 µm internal diameter.
   1. In the case that the HPLC is not programmed to automate column equilibration before sample loading, include (4) 90%-4% MPB over 1 min and (5) isocratic 4% MPB over 10 min.
3. Program the MS acquisition method.
   1. Ensure that the instrument performs one full MS scan at the beginning of each duty cycle. High-resolution instrumentation (e.g., orbitrap or time-of-flight analyzers) is recommended, due to the mass accuracy that can be utilized during signal extraction. However, low-resolution instrumentation can also be utilized as previously described^27,28.
   2. Ensure that the full MS scan is followed by 16 MS/MS scan events, each one with an isolation width of 50 m/z, spanning the m/z range of 300-1100. For example, the first scan should isolate signals at 300-350 m/z, the second at 350-400 m/z, etc. If possible, acquire MS/MS scans in high resolution also, but lower resolution compared to the full MS scan is sufficient (due to the smaller masses of fragment ions compared to intact ions).
   3. The HPLC method will generate chromatographic signals with peak widths of ~3-40 s; to ensure proper signal quantification, ensure that the mass spectrometer performs at least 10 duty cycles per chromatographic peak (i.e., a duty cycle of 3 s or faster).
   4. If using a trapping-style mass analyzer (orbitrap, ion trap), ensure that the ion injection time limit is set to <200 ms; for other analyzers (quadrupole, time-of-flight), this is not an issue due to their faster scan time. A preliminary test might be required.
      NOTE: More details on MS methods for histone peptide analysis can be found in the following references^27,28,29.
4. Resuspend sample in 10 µL of MPA, which corresponds to ~1 µg/µL of digested histone sample. The exact loading amount is not critical (also not trivial to assess) if all samples in the batch are loaded using similar dilutions and volumes.
5. Load 1 µL of sample onto the HPLC column.
6. Run the LC-MS/MS method programmed in steps 10.1-10.3.

11. Data analysis

1. Import the MS raw data files into software designed to perform peak area integration.
   NOTE: EpiProfile^30,31 is used in the current study and is generally recommended, as it is optimized for reliable peak area extraction of known histone peptides. However, other freely available software for extracted ion chromatography such as Skyline^32,33 are suitable.
2. Calculate the relative abundance of a given (un)modified peptide as the area of a single peptide divided by the total area of the peptide in all its modified forms. Software such as EpiProfile^30,31 already contains libraries of peptides for signal extraction. Otherwise, generate a library of peptides of interest either manually or via peptide identification using routine proteomics pipelines.

Results

In this protocol, HepG2/C3A spheroids were treated with 20 mM NaBut and 10 mM NaSuc, both of which affected global levels of histone PTMs (Figure 3A). Histone PTMs were then identified and quantified at the single residue level via MS/MS acquisition (Figure 3B).

When samples are run in replicates, statistical analysis can be performed to assess the fold change enrichment of a PTM between samples, as well as the reproducibilit...

Discussion

The analysis of histone PTMs is fundamentally different from the typical proteomics analysis pipeline. Most histone PTMs still have enigmatic biological functions; as a result, annotations such as Gene Ontology or pathway databases are not available. Several resources exist that associate histone modifications with the enzyme responsible for their catalysis or proteins containing domains that bind these PTMs (e.g., HISTome³⁶). As well, it is possible to speculate on the overall state of the chroma...

Materials

Name	Company	Catalog Number	Comments
0.05% trypsin-EDTA solution	Gibco	25300054
0.5-20 µL pipet tips	BRAND	13-889-172 (Fisher Scientific)
1.5 mL microcentrifuge tubes	Bio-Rad	2239480
10 µL multi-channel pipette	BRAND	BR7059000 (Millipore Sigma)
10 mL syringe	Henke Sass Wolf	14-817-31 (Fisher Scientific)	Luer lock tip, graduated to 12 mL
10, 20, 200, and 1000 µL single-channel pipettes	Eppendorf	14-285-904 (Fisher Scientific)
1000 µL pipet tips	Rainin	30389164
18 G syringe needle	Air-Tite	14-817-100 (Fisher Scientific)	3" length, 0.05" diameter
200 µL multi-channel pipette	Corning	4082
2-200 µL pipet tips	BRAND	Z740118 (Millipore Sigma)
24-well ultra-low attachment microplate	Corning	07-200-602
75 cm2  U-shaped cell culture flask	Corning	461464U	Untreated, with vent cap
96-well skirted plate	Axygen	PCR-96-FS-C (Corning)
Acetone	Fisher Scientific	A949-1	Acetone should be used cold
Ammonium bicarbonate (NH4HCO3)	Sigma-Aldrich	A6141-25G
Ammonium hydroxide solution	Fisher Scientific	AC423300250
Cell culture grade water	Corning	25-055-CV
ClinoReactor	CelVivo	10004-12	Bioreactor for 3D cell culture
ClinoStar	CelVivo	N/A	Clinostat CO2 incubator for 3D cell culture
Control unit	CelVivo	N/A	Tablet for ClinoStar settings
Dulbecco's Modified Eagle's Medium (DMEM)	Corning	17-205-CV	1X solution with 4.5 g/L glucose and sodium pyruvate, without L-glutamine and phenol red
Fetal bovine serum (FBS)	Corning	35-010-CV
Formic acid	Thermo Scientific	28905
Fume hood	Mott	N/A	Model 7121000
Glass Pasteur pipette	Fisher Scientific	13-678-8B	9", cotton-plugged, borosilicate glass, non-sterile
Glutagro supplement	Corning	25-015-CI	200 mM L-ananyl-L-glutamine
Hank’s Balanced Salt Solution (HBSS)	Corning	21-022-CV	1X solution without calcium, magnesium, and phenol red
HPLC grade acetonitrile	Fisher Scientific	A955-4
HPLC grade water	Fisher Scientific	W6-1
Hydrochloric acid (HCl)	Fisher Scientific	A481-212
Ice	N/A	N/A
MEM non-essential amino acids	Corning	25-025-CI	100X solution
Oasis HLB resin	Waters	186007549	Hydrophilic-Lipophilic-Balanced (HLB) Resin with 30µm particle size
Orbitrap Fusion Lumos Tribrid mass spectrometer	Thermo Fisher Scientific	IQLAAEGAAPFADBMBHQ	High resolution mass spectrometer
Oro-Flex I polypropylene filter plate	Orochem	OF1100	96-well polypropylene filter plate w/ 10 µM PE frit
Penicillin-Streptomycin	Corning	30-002-CI	100X solution
pH paper	Hydrion	Z111848 (Sigma-Aldrich)	0-13 pH test paper
Pipette gun	Eppendorf	Z666467 (Millipore Sigma)
Polymicro capillary	Molex	50-110-7740 (Fisher Scientific)	Flexible fused silica capillary tubing with polymide coating, 75 µM ID x 363 µM OD
Polystyrene 10 mL serological pipets, sterile	Fisher Scientific	1367549
Propionic anhydride	Sigma-Aldrich	240311-50G
Refrigerated centrifuge	Thermo Scientific	75-217-420
Reprosil-Pur resin	MSWIL	R13.AQ.0003	120 Å pore size, C18-AQ phase, 3 µM bead size
Rotator	Clay Adams	25477 (American Laboratory Trading)	Nutator Mixer 1105
Sequencing grade modified trypsin	Promega	V5111
Sodium butyrate	Thermo Scientific	A11079
Sodium succinate dibasic	Sigma-Aldrich	14160-100G
SpeedVac vacuum concentrator (1.5 mL microcentrifuge tubes)	Savant	20249 (American Laboratory Trading)
SpeedVac vacuum concentrator (96-well)	Thermo Scientific	15308325	Savant SPD1010
Sterile hood	Thermo Scientific	1375	Class II, Type A2
Sulfuric acid (H2SO4)	Fisher Scientific	02-004-375	Baker Analyzed ACS reagent
Tissue-culture treated 100 mm x 20 mm dish	Fisher Scientific	08-772-23
Trichloroacetic acid (TCA)	Thermo Scientific	AC421451000	Resuspend 100% w/v in HPLC grade water
Trifluoroacetic acid (TFA)	Fisher Scientific	PI28904	Sequencing grade
Vacuum manifold 96-well	Millipore	MAVM0960R
Vortex	Sigma-Aldrich	Z258415
Water bath	Fisher Scientific	FSGPD10
Wide bore pipet tips 1000 µL	Axygen	14-222-703 (Fisher Scientific)
Wide bore pipet tips 200 µL	Axygen	14-222-730 (Fisher Scientific)