Name: global level quantification of histone post-translational modifications in a 3d cell culture model of hepatic tissue
Uploaded: 2022-05-05T14:00:00
Description: Watch this Scientific Journal Video about Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue at JoVE.com

The Sidoli lab gratefully acknowledges the Leukemia Research Foundation (Hollis Brownstein New Investigator Research Grant), AFAR (Sagol Network GerOmics award), Deerfield (Xseed award), Relay Therapeutics, Merck, and the NIH Office of the Director (1S10OD030286-01).

1. Preparation of buffers and reagents
<ol>
	<li>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&#39;s Modified Eagle&#39;s Medium (DMEM, containing 4.5 g/L glucose). Growth media is stored at 4 &#176;C for a maximum of 2 weeks.</li>
	<li>200 mM sodium butyrate (NaBut) solution: To prepare 10 mL, resuspend 220.18 mg of NaBut in 10 mL of ddH2O...

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...

Since the late 19th century, cell culture systems have been used as a model to study the growth and development of cells outside of the human body1,2. Their use has also been extended to study how tissues and organs function in both healthy and diseased contexts1,3. 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 surface2,4. 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 vivo1,2. 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&#160;(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 discussion10,11,12,13, 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 &#34;histone code&#34; 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 &#34;histone code&#34;.
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 combinations18,19,20. MS is more suitable for high-throughput and unbiased identification and quantification of single and co-existing protein modifications, in particular histone proteins18,19,20. 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 culture24,25. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA solution</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5-20 &#181;L pipet tips</td>
			<td>BRAND</td>
			<td>13-889-172 (Fisher Scientific)</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Bio-Rad</td>
			<td>2239480</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L multi-channel pipette</td>
			<td>BRAND</td>
			<td>BR7059000 (Millipore Sigma)</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Henke Sass Wolf</td>
			<td>14-817-31 (Fisher Scientific)</td>
			<td>Luer lock tip, graduated to 12 mL</td>
		</tr>
		<tr>
			<td>10, 20, 200, and 1000 &#181;L single-channel pipettes</td>
			<td>Eppendorf</td>
			<td>14-285-904 (Fisher Scientific)</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L pipet tips</td>
			<td>Rainin</td>
			<td>30389164</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G syringe needle</td>
			<td>Air-Tite</td>
			<td>14-817-100 (Fisher Scientific)</td>
			<td>3&#34; length, 0.05&#34; diameter</td>
		</tr>
		<tr>
			<td>200 &#181;L multi-channel pipette</td>
			<td>Corning</td>
			<td>4082</td>
			<td></td>
		</tr>
		<tr>
			<td>2-200 &#181;L pipet tips</td>
			<td>BRAND</td>
			<td>Z740118 (Millipore Sigma)</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well ultra-low attachment microplate</td>
			<td>Corning</td>
			<td>07-200-602</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2&#160; U-shaped cell culture flask</td>
			<td>Corning</td>
			<td>461464U</td>
			<td>Untreated, with vent cap</td>
		</tr>
		<tr>
			<td>96-well skirted plate</td>
			<td>Axygen</td>
			<td>PCR-96-FS-C (Corning)</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A949-1</td>
			<td>Acetone should be used cold</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate (NH4HCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>A6141-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Fisher Scientific</td>
			<td>AC423300250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Corning</td>
			<td>25-055-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>ClinoReactor</td>
			<td>CelVivo</td>
			<td>10004-12</td>
			<td>Bioreactor for 3D cell culture</td>
		</tr>
		<tr>
			<td>ClinoStar</td>
			<td>CelVivo</td>
			<td>N/A</td>
			<td>Clinostat CO2 incubator for 3D cell culture</td>
		</tr>
		<tr>
			<td>Control unit</td>
			<td>CelVivo</td>
			<td>N/A</td>
			<td>Tablet for ClinoStar settings</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>17-205-CV</td>
			<td>1X solution with 4.5 g/L glucose and sodium pyruvate, without L-glutamine and phenol red</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td>Mott</td>
			<td>N/A</td>
			<td>Model 7121000</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-8B</td>
			<td>9&#34;, cotton-plugged, borosilicate glass, non-sterile</td>
		</tr>
		<tr>
			<td>Glutagro supplement</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td>200 mM L-ananyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td>1X solution without calcium, magnesium, and phenol red</td>
		</tr>
		<tr>
			<td>HPLC grade acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W6-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A481-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td>100X solution</td>
		</tr>
		<tr>
			<td>Oasis HLB resin</td>
			<td>Waters</td>
			<td>186007549</td>
			<td>Hydrophilic-Lipophilic-Balanced (HLB) Resin with 30&#181;m particle size</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGAAPFADBMBHQ</td>
			<td>High resolution mass spectrometer</td>
		</tr>
		<tr>
			<td>Oro-Flex I polypropylene filter plate</td>
			<td>Orochem</td>
			<td>OF1100</td>
			<td>96-well polypropylene filter plate w/ 10 &#181;M PE frit</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>100X solution</td>
		</tr>
		<tr>
			<td>pH paper</td>
			<td>Hydrion</td>
			<td>Z111848 (Sigma-Aldrich)</td>
			<td>0-13 pH test paper</td>
		</tr>
		<tr>
			<td>Pipette gun</td>
			<td>Eppendorf</td>
			<td>Z666467 (Millipore Sigma)</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymicro capillary</td>
			<td>Molex</td>
			<td>50-110-7740 (Fisher Scientific)</td>
			<td>Flexible fused silica capillary tubing with polymide coating, 75 &#181;M ID x 363 &#181;M OD</td>
		</tr>
		<tr>
			<td>Polystyrene 10 mL serological pipets, sterile</td>
			<td>Fisher Scientific</td>
			<td>1367549</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>240311-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-217-420</td>
			<td></td>
		</tr>
		<tr>
			<td>Reprosil-Pur resin</td>
			<td>MSWIL</td>
			<td>R13.AQ.0003</td>
			<td>120 &#197; pore size, C18-AQ phase, 3 &#181;M bead size</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Clay Adams</td>
			<td>25477 (American Laboratory Trading)</td>
			<td>Nutator Mixer 1105</td>
		</tr>
		<tr>
			<td>Sequencing grade modified trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Thermo Scientific</td>
			<td>A11079</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>14160-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator (1.5 mL microcentrifuge tubes)</td>
			<td>Savant</td>
			<td>20249 (American Laboratory Trading)</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator (96-well)</td>
			<td>Thermo Scientific</td>
			<td>15308325</td>
			<td>Savant SPD1010</td>
		</tr>
		<tr>
			<td>Sterile hood</td>
			<td>Thermo Scientific</td>
			<td>1375</td>
			<td>Class II, Type A2</td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4)</td>
			<td>Fisher Scientific</td>
			<td>02-004-375</td>
			<td>Baker Analyzed ACS reagent</td>
		</tr>
		<tr>
			<td>Tissue-culture treated 100 mm x 20 mm dish</td>
			<td>Fisher Scientific</td>
			<td>08-772-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Thermo Scientific</td>
			<td>AC421451000</td>
			<td>Resuspend 100% w/v in HPLC grade water</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Fisher Scientific</td>
			<td>PI28904</td>
			<td>Sequencing grade</td>
		</tr>
		<tr>
			<td>Vacuum manifold 96-well</td>
			<td>Millipore</td>
			<td>MAVM0960R</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Sigma-Aldrich</td>
			<td>Z258415</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>FSGPD10</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide bore pipet tips 1000 &#181;L</td>
			<td>Axygen</td>
			<td>14-222-703 (Fisher Scientific)</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide bore pipet tips 200 &#181;L</td>
			<td>Axygen</td>
			<td>14-222-730 (Fisher Scientific)</td>
			<td></td>
		</tr>
	</tbody>
</table>

global level quantification of histone post-translational modifications in a 3d cell culture model of hepatic tissue

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.

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...

Watch this Scientific Journal Video about Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue at JoVE.com

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

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.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

Global chromatin dynamics have been shown to be an important mediator of several disease states, such as cancer and aging. Our protocol provides a physiologically relevant model to study this process. Standard laboratory analysis of histone post-translational modifications or PTMS is usually limited to probing for a single PTM at a time using antibody-based assays.Liquid chromatography mass spectrometry analysis of histone PTMS allows us to measure the abundance of hundreds of PTMS in a single experiment. Demonstrating the procedure will be Stephanie Stransky, Ronald Cutler, and Julie Kim, a postdoc, grad student, and research tech, respectively, from the lab. First, using standard growth media, grow the cells as a monolayer until they are 80%confluent.Then wash the cells with HBSS. Add five milliliters of 0.05%trypsin-EDTA diluted in HBSS. Incubate the cells for five minutes at 37 degrees Celsius with 5%carbon dioxide.Use a microscope to check cell detachment. After counting the cells, dilute the cell suspension with fresh growth media. Add 0.5 milliliters of growth media to equilibrate an ultra low attachment 24-well round bottom plate with multiple micro-wells in each.Then centrifuge the plates at 3000 x g to remove air bubbles from the surface of the plate. Now transfer the previously made cell suspension to the 24-well plate and centrifuge for three minutes at 120 x g. Check the cells in the 24-well plate and then incubate the plates at 37 degrees Celsius with 5%carbon dioxide for 24 hours for spheroid formation.Meanwhile, to equilibrate the bioreactor, add 25 milliliters of sterile water to the humidity chamber and nine milliliters of growth media to the cell chamber. Incubate the bioreactor in a rotating clinostat incubator for 24 hours at 37 degrees Celsius with 5%carbon dioxide. Detach the spheroids from the ultra low attachment 24-well plate by gently pipetting using one milliliter wide bore tips, and transfer them to a tissue culture dish.To select sufficiently forms spheroids, check the quality of spheroids under a microscope. Good quality spheroids have uniform size, compactness, and roundness. Fill the equilibrated bioreactor with five milliliters of fresh growth media, and transfer the spheroids into it.Then fill the bioreactor completely with fresh growth media and put the bioreactor in the clinostat incubator at 10 to 11 RPM. Every two to three days, exchange growth media by replacing 10 milliliters of old media with fresh media. As this spheroids increase in size and number, increase the rotation speed of the incubator.After obtaining the spheroid pellet, add five volumes of cold 0.2 molar sulfuric acid to the pellet and pipette up and down to disrupt the pellet and release histones. Once the supernatant is obtained after centrifugation, add cold concentrated trichloroacetic acid, such that the final concentration becomes 25 to 30%volume by volume. And mix it by inverting the tube a few times and centrifuge as described in the manuscript.After discarding the supernatant, using a glass pasture pipette, wash the pellet and the walls of the tube with approximately 500 microliters of cold acetone and 0.1%hydrochloric acid, and then centrifuge at 3, 400 x g for five minutes at four degrees Celsius. Then flip the tube to discard the supernatant. Using a glass pasture pipette, add 500 microliters of 100%cold acetone to wash the pellet and centrifuge as demonstrated previously.After discarding the supernatant, completely remove acetone by pipetting carefully, and let the sample dry with an open lid for about 20 minutes. Resuspend the pellet in 20 microliters of 15 to 20%of acetonitrile in a 100 millimolar ammonium bicarbonate of pH 8. After vortexing, spin down the suspension at 1000 x g for 30 seconds.For eight or more samples, transfer each re suspended sample to a 96-well plate. Then, in a hood, add two microliters of propionic anhydride and mix by pipetting five times. Quickly add 10 microliters of ammonium hydroxide and mix by pipetting five times.Mix HLB resin on a magnetic stir plate. Then add 70 microliters of the HLB suspension to each well of a 96-well filter plate on a 96-well collection plate. After discarding the flow through, wash the resin with 100 microliters of 0.1%trifluoroacetic acid.After resuspending each sample in 100 microliters of 0.1%trifluoroacetic acid, load each sample in each well and prevent splashing using a vacuum. After discarding the flow through, wash the samples with 100 microliters of 0.1%trifluoroacetic acid, and put the filter plate on a new collection plate. Next, add 60 microliters of 60%acetonitrile in 0.1%trifluoroacetic acid to each well and prevent splashing using vacuum.Collect the flow through and dry in a speed vacuum. Load the dried collection plate into the HPLC and run the LC/MS/MS method as described in the manuscript. In this study, the relative abundance of common histone post-translational modifications was observed in C3A hepatocytes grown as three dimensional spheroids.Post-translational modifications could also be observed on a single residue in peptides generated from histone proteins. Treatment of the spheroids with sodium butyrate increased the relative abundance of histone acetylation, whereas that with sodium succinate enhanced histone residue succinylation. The method also demonstrated the distribution pattern of histone acetylation after sodium butyrate treatment and that of histone succinylation after treatment with sodium succinate.The result also showed commonatorial patterns of different histone modifications. Sodium butyrate treatments significantly enhanced the acetylation of the lysine residue 14 on a peptide generated from the histone protein H3, only when its ninth lysine residue had two methyl groups, but not one or three methyl groups. The relative abundances of individual modifications and various combinations of post-translational modifications in the H3-derived peptide were also calculated.Combinatorial patterns of post-translational modifications after sodium butyrate treatment were also observed in a peptide generated from histone protein H4.Here also, sodium butyrate treatment resulted in a significant increase in acetylation. Critical attention should be placed on keeping the spheroids on the bench as little as possible and in a desalting step to avoid sample spilling. This workflow can be used to explore chromatin in solid tissues using a more physiological model than fast replicating flat cell cultures.

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Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...