The work was supported by PRIN2022, DS, CF, and AC were supported by PRIN2022, and MLC was supported by PhD program in Molecular Oncology, UMG PhD program school. The work was also supported by Project PNRR INSIDE CUP: B83C22003920001

1. Histone extraction
<ol>
	<li>Perform histone extraction from at least 1 x 106 cells, which are 80% confluent. For this experiment, we used MCF-10A cell lines from ATCC. After plating, wash cells thrice with 1 mL of phosphate-buffered saline (PBS) 1x. Perform all the steps on ice.</li>
	<li>Prepare a cell lysis buffer containing 10 mM Tris-HCl pH 8.0, 1 mM KCl, and 1.5 mM MgCl2. Add 1 mM dithioerythritol and protease and phosphatase inhibitors just before use.</li>
	<li>Add 800 &#181;L of cell lysis buffer to the cell-containing Petri dish, scrape, and collect the cells in a 1.5 mL tube, then incubate on a rotator at 300 rpm, 4 &#176;C for 1 h.</li>
	<li>Centrifugate at 10,000 x g, 4 &#176;C, for 10 min in a precooled microcentrifuge with a rotator for 1.5 mL tubes.</li>
	<li>Wash the pellet with 400 &#181;L of cell lysis buffer and centrifuge at 10,000 x g, 4 &#176;C, for 10 min. Discard the supernatant. Repeat the washing step 3x. 
	NOTE: Use a precooled microcentrifuge (4 &#176;C) with a rotator for 1.5 ml tubes for all the steps. Handle the pellet carefully to avoid loss of material.</li>
	<li>Resuspend the pellet in 640 &#181;L of 0.2 M H2SO4 and incubate on a rotator at 300 rpm, at 4 &#176;C, for 16 h. 
	CAUTION: Sulfuric acid is a hazardous chemical; please use it under a chemical fume hood.</li>
	<li>Centrifuge at 16,000 x g, 4 &#176;C, for 10 min. Transfer the supernatant to a fresh tube and store it until the following steps. Discard the nuclear debris.</li>
	<li>Precipitate histones by adding 212 &#181;L of Trichloroacetic acid, drop by drop (the final concentration of TCA is 33%). Mix the solution by inverting the tube 10x. Incubate on ice, in the dark, for 16 h. 
	CAUTION: TCA is a hazardous chemical; use under a chemical fume hood.</li>
	<li>Pellet histones by spinning in a precooled centrifuge at 16,000 x g for 10 min at 4 &#176;C. Carefully remove the supernatant and wash the pellet 3x with 200 &#181;L of cold acetone.</li>
	<li>Resuspend the pellet with cold acetone, then sonicate high power 2x with 3 cycles of 30 s on/off, alternating with a 10 min pause on ice. 
	NOTE: The procedure was done using an ultrasonicator. This step is essential to eliminate histone-wrapped DNA.</li>
	<li>Centrifuge at 16,000 x g for 10 min, discard acetone, and resuspend the sample in 100 &#181;L of double-distilled water. Determine protein concentration using the Bradford assay method, according to the manufacturer&#39;s instructions33. 
	NOTE: After centrifugation, the pellet sometimes appears white or translucent. A white and opaque precipitate could indicate the presence of salt contaminants that might impair the 2D separation. If so, repeat the acetone wash steps until the pellet becomes translucid.</li>
	<li>Lyophilize 70 &#181;g of histones, according to the manufacturer&#39;s instructions. Set the lyophilizer at 30 &#176;C for 60 min. Check the tube volume and reiterate the step at 30 &#176;C until the sample is completely lyophilized (generally, 90 min is necessary to completely lyophilize the sample). After 60 min, check the volume every 10 min since the process is sample-dependent. 
	NOTE: If the amount of histone proteins is low, start from 2 x 106 cells. In this case, use the same volume of reagents used for 1 x 106 cells. Check the purity of all the chemicals used during the procedure; all the reagents must be HPLC grade.</li>
</ol>
2. Mini 2D gel triton-acid-urea (TAU) electrophoresis
<ol>
	<li>Prepare a mini-TAU gel for a mini vertical polyacrylamide gel electrophoresis system as described below.
	<ol>
		<li>Prepare separating gel: 6 M Urea, 15% acrylamide/bisacrylamide (29:1), 5% glacial acid acetic, 0.25% Triton X-100 (v/v), 0.4% TEMED (v/v), 0.1% APS (p/v). To prepare the solution, dissolve urea in acrylamide/bis and 1 mL of ultrapure water with gentle agitation at room temperature; add the other components and adjust the volume. For a mini-TAU gel, prepare 10 mL of solution. Fill the preset gel plates with the solution, avoiding the formation of air bubbles just 1.5 cm from the top. Cover the solution with 1 mL of isopropanol to level out the gel.</li>
		<li>Prepare stacking gel: 6 M Urea, 6% acrylamide/bisacrylamide (29:1), 5% glacial acid acetic, 0.25% Triton X-100 (v/v), 0.4% TEMED (v/v), 0.1% APS (p/v). To prepare the solution, dissolve urea in acrylamide/bis and 1 mL of ultrapure water; add the other components and adjust to volume. Fill gel plates with stacking solution, place a 10-well gel comb between plates, and insert it until the gel solution comes halfway up the teeth of the comb. Allow the gel to polymerize 16 h at 4 &#176;C. 
		NOTE: Carefully insert the comb to ensure the regular formation of wells.</li>
	</ol>
	</li>
	<li>Prepare 100 &#181;L of TAU sample buffer by dissolving 6 M Urea, 5% glacial acid acetic, and double-distilled water. Dissolve the lyophilized sample prepared in 30 &#181;L of TAU sample buffer.</li>
	<li>Fill the mini cassette with TAU running buffer, 5% acetic acid final concentration. Rinse the wells of the TAU-gel with a syringe filled with running buffer and load the sample in the well using a micropipette.</li>
	<li>Connect the electrophoretic chamber to the power supply, with the positive (red) electrode placed at the bottom. Pay attention, when IP &#62; pH, proteins are positively charged, so the migration is from positive to negative electrode. Run the gel first at 30 V for 70 min, then at the constant voltage of 25 V for 17 h. 
	NOTE: During this time, occasionally switch off the power supply and rinse the gel wells using a disposable syringe. Pay attention to removing any bubbles from the bottom surface of the gel using a syringe to avoid the loss of sample and the presence of a smearing in the resolved proteins.</li>
	<li>Remove the gel from the gel plates and cut the line of interest with a clean scalpel. Carefully handle the gel to avoid gel breakage. Since the line of interest is colorless, load the sample in well number 1 to exactly identify and cut the lane of interest. 
	NOTE: To check the separation on mini-TAU gel, prepare and run the sample in duplicate. One of these gels can be stained using Coomassie Brilliant Blue. The pattern of separation is reported in Figure 1A.</li>
	<li>Wash the lane with 15 mL of 0.5 M Tris-HCl pH 8.8 for 15 min.</li>
	<li>Prepare 15 mL of equilibration buffer 1 using 6 M Urea, 0.5 M Tris-HCl, pH 6.8, 30% glycerol, 20% SDS, and 2% Dithioerythritol. Prepare 15 mL of equilibration buffer 2, using 6 M Urea, 0.5 M Tris-HCl, pH 6.8, 30% glycerol, 20% SDS, and 2% Iodoacetamide. Prepare the equilibration buffer 1 and 2 just before use; store the equilibration buffer 2 in the dark.</li>
	<li>Incubate the gel lane, under gentle agitation, in 10 mL of equilibration buffer 1 for 15 min, then decant. Incubate the gel lane, under gentle agitation, with 10 mL of equilibration buffer 2 for 15 min in the dark, then decant.</li>
	<li>Prepare a resolving SDS 15% acrylamide gel by following Table 1. Set up the gel plate and fill it with the resolving gel solution. Fill the solution until it is 1.5 cm from the end of the long gel plate. Put 1 mL of isopropanol on the surface to level the gel and allow it to polymerize.</li>
	<li>After gel polymerization, decant the isopropanol and dry the well with 3MM Chr Cellulose Chromatography Paper. Insert the resulting lane from step 8 into the gel well.</li>
	<li>Seal the lane to the resolving gel with a sealing solution of 0.5% low melting point agarose in 1x Tris/glycine/SDS buffer and 5 &#181;L of 0.003% bromophenol blue. Allow agarose gel polymerization for 30 min. 
	NOTE: Avoid the formation of air bubbles during agarose sealing to prevent gel smearing.</li>
	<li>Put the gel plates in the cassette and fill with 1x Tris/glycine/SDS buffer. Run the gel at 120 V until the bromophenol blue dye reaches the bottom. Gently remove the gel from the plates, discard the excess of agarose, and silver stain the gel using a procedure compatible with mass spectrometry or blot for immunological assay.</li>
</ol>
3. Silver staining 32
<ol>
	<li>Prepare the following reagents - Fixative: 50% methanol, 5% acetic acid, 45% deionized water; Wash solution: 50% methanol, 50% deionized water; Sensitizer: 0.02% Na2S2O3 5H2O in deionized water; Silver reagent: 0.1% silver nitrate in cold (4&#176;C) deionized water; Developer: 0.04% Formalin and 2% Na2CO3, Formaldehyde (37%) (add just before use) 0.4 mL deionized water and fill up to 1000 mL; Stop-reagent: 5% acetic acid; Gel storage solution: 1% acetic acid. 
	NOTE: Since chemicals used for staining are hazardous, perform all the steps under a chemical fume hood.</li>
	<li>Follow the procedure in Table 2 with the following consideration. Perform all incubations with gentle agitation. Prepare the stop reagent before starting the developing step; this will ensure gel development with the opportune intensity. During the developing step, spots will appear as yellow signals that will become light brown at the end of the incubation. The intensity of the different spots will depend on the amount of histone proteoforms. The gel is ready for spot excision and in-gel digestion (Figure 1B)</li>
</ol>
4. In-gel digestion
<ol>
	<li>Prepare the following solutions: 50 mM ammonium bicarbonate (NH4HCO3)/50% (v/v) acetonitrile; 10 mM dithiothreitol (DTT) in 50 mM NH4HCO3; 55 mM iodoacetamide (IAA) in 50 mM NH4HCO3; 0.1% TFA; 50 mM NH4HCO3, pH 8; HPLC grade Acetonitrile; 1 mL silicon tubes. 
	NOTE: To circumvent human keratin contamination, from hair or skin, use clean gloves and work in a clean area.</li>
	<li>Excise histone spots of interest from the stained gel using a clean scalpel and place them into a fresh silicon tube (1.5 mL). Take care to excise a single spot and put it in a single tube to avoid isoform cross-contamination.</li>
	<li>Add to each gel spot 250 &#181;L of 50 mM NH4HCO3/50% acetonitrile and incubate under gentle agitation in a thermomixer at room temperature. Decant the solution and repeat this step 3x. Gel pieces will appear transparent.</li>
	<li>Dehydrate gel spots by adding acetonitrile (200 &#181;L). At this stage the gel pieces will take an opaque-white color.</li>
	<li>Decant acetonitrile and let air-dry for 10 min. The gel piece will appear dried in the tube. Reduction and alkylation are not essential since histone proteins have been reduced and alkylated during the equilibration step.</li>
	<li>Rehydrate gel spots in 20 &#181;L of trypsin solution (0.1 mM HCl) or a volume sufficient to cover the gel pieces. Place the tubes in the thermomixer and incubate, under gentle agitation, at 37 &#176;C for a minimum of 4 h until 16 h.</li>
	<li>Transfer the supernatant containing the tryptic peptides to a fresh tube. Add 50 &#181;L of extraction solution (60% acetonitrile, 1% TFA) to the gel pieces and sonicate 2x in an ultrasonic water bath for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant, containing additional tryptic peptides, to the fresh tube. Use a lyophilizer to dry the pooled extracted peptides. Set the lyophilizer at 30 &#176;C and dry the solution; a lyophilized pellet will appear in the tube. 
	NOTE: This step is volume-dependent; generally, 1 h is enough, but check the volume every 10 min until the sample is completely dried.</li>
	<li>Add 15 &#181;L of 0.1 % TFA to each tube and gently incubate in a thermomixer at 25 &#176;C. Transfer the supernatant to the vial for LC-MS/MS analysis.</li>
</ol>
5. 2DTAU western blot
<ol>
	<li>Prepare the transfer buffer, 250 mM Tris base, 92 mM glycine, and 10% methanol using double-distilled water.</li>
	<li>Cut the nitrocellulose membrane and the filter paper to the dimensions of the gel.</li>
	<li>Equilibrate the gel and soak the membrane, filter paper, and filter papers in the transfer buffer for 15 min. Use either Nitrocellulose or PVDF membranes. If using PVDF (Polyvinylidene difluoride) membranes, activate them by soaking in 100% MetOH.</li>
	<li>Prepare the gel sandwich in the cassette for a Semi-dry transfer as follows: Negative electrode/Filter-Paper/Gel/Nitrocellulose-membrane/Filter-paper/Positive electrode. Remove any air bubbles between gel and membrane to ensure good results.</li>
	<li>Place the cassette in the Semi-dry unit and run the blot at 25 V for 30 min. At the end, disassemble the blotting sandwich and store the membrane for the immunological assay. Check the transfer efficiency with Ponceau staining. Mark the membrane with a sign at the top right to preserve the right filter orientation.</li>
	<li>Incubate with primary and secondary antibodies depending on the experiment (Figure 2).</li>
</ol>

2D Gel Separation for Histone Isoform Enrichment and PTM Characterization

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+the+human+proteome+reaches+a+major+milestone:+&gt;90%+of+predicted+human+proteins+now+credibly+detected,+according+to+the+HUPO+human+proteome+project.">Research on the human proteome reaches a major milestone: &gt;90% of predicted human proteins now credibly detected, according to the HUPO human proteome project.</a> J Proteome Res. 19 (12), 4735-4746 (2020).</li><li>Noberini, R., Robusti, G., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+characterization+of+histones+in+clinical+samples:+applications,+progress,+and+challenges.">Mass spectrometry-based characterization of histones in clinical samples: applications, progress, and challenges.</a> FEBS J. 289 (5), 1191-1213 (2022).</li><li>Starkova, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+profile+of+post-translational+modifications+of+histone+H1+in+chromatin+of+mouse+embryonic+stem+cells.">The profile of post-translational modifications of histone H1 in chromatin of mouse embryonic stem cells.</a> Acta Naturae. 11 (2), 82-91 (2019).</li><li>Garc&#237;a-Gim&#233;nez, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+carbonylation+occurs+in+proliferating+cells.">Histone carbonylation occurs in proliferating cells.</a> Free Radic Biol Med. 52 (8), 1453-1464 (2012).</li><li>Garc&#237;a-Saura, A. G., Sch&#252;ler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP10+multi-site+auto-+and+histone+MARylation+visualized+by+acid-urea+gel+electrophoresis.">PARP10 multi-site auto- and histone MARylation visualized by acid-urea gel electrophoresis.</a> Cells. 10, 654 (2021).</li></ol>

The authors declare no conflicts of interest.

In the past decade, cancer patient therapy has been revolutionized by identifying various molecular alterations that drive cancer development and progression. The most significant advancement in modern oncology is the development of therapy based on comprehensive molecular analysis. Technological advances in genomics and proteomics have played a crucial role in profiling specific biomarkers for early detection, surveillance, prognosis, and drug monitoring. Proteomics is defined as the analysis of the complete protein com...

Cancer onset, progression, and chemoresistance are related to genetic and epigenetic events, including histone PTMs. Histones undergo a variety of PTMs that regulate chromatin dynamics and dictate cell fate1.
The nucleosome is the functional unit of chromatin and is a molecular complex of DNA and proteins that allows DNA to be packaged into the nucleus. It consists of four pairs of histones (H3, H4, H2A, and H2B) that constitute the nucleosome core, as well as the linker histone H1, which helps stabilize the chromatin structure. The histones amino (N)-terminal tails protrude from their globular core domains and undergo specific post-translational modifications that affect the chromatin landscape2.
So far, at least 23 classes of histone PTMs are known. These PTMs are covalent modifications that mainly consist of methylation, acetylation, phosphorylation, glycosylation, ubiquitylation, SUMOylation, and ADP-ribosylation3. In addition, a tight correlation between epigenome homeostasis and metabolic rewiring has recently been proposed, which explains the exponential growth of the possible number of histones PTMs4,5,6,7,8,9,10,11,12,13. Histone PTMs, deposited, erased, and transduced by modifier enzymes, are named respectively as writers, erasers, and readers. They affect the physical and chemical properties of chromatin structure, impacting gene expression profile, including the expression of tumor suppressor genes and oncogenes14,15,16.
The number of histones proteoforms increases when incorporated into the nucleosome as histone variants. These variants are non-allelic isoforms of canonical histones, and they add complexity by altering the chemical and physical properties of chromatin. In comparison to canonical histones, histone variants have functional domains that undergo unique post-translational modifications or interact with specific chromatin components, impacting the dynamics and stability of the nucleosome17.
In cancer cells, the deregulation of histone PTMs and variants affects gene expression profile, leading to abnormal cellular proliferation, invasion, metastasis, and the activation of drug resistance pathways3,18,19,20. Mutations within histones or chromatin remodeling complexes can alter cell phenotype, contributing to neoplastic transformation. The accumulation of epigenetic alterations, such as histone methylation, is a key factor in activating drug resistance pathways21,22,23. Mutations within the SWI/SNF complex, which consists of 15 subunits encoded by 29 genes, impact over 20% of human cancers across many tumor types. In the past decade, epigenetic-based drug strategies (epi-drugs) have been developed, and many drugs targeting cancer with abnormal histone modification patterns have been approved by the US Food and Drug Administration (FDA)23.
The analysis of histone proteins is quite challenging, making it a demanding area of study. Recently, mass spectrometry methods have become the preferred way for characterizing and studying histone proteoforms. However, the drastic extraction conditions, in combination with the poor solubility of some variants and the high sequence homology, make their analysis challenging.
In the past decade, several mass spectrometry-based methods have been developed for histone profiling and investigation24,25,26,27. In this study, we present a cost-effective and reliable method to create a two-dimensional map of histone proteoforms, which can enhance the chances of discovering new post-translational modifications.
2D TAU electrophoresis combines separation in acidic conditions with SDS PAGE. In the first dimension, the medium&#39;s acidic condition gives a net positive charge to basic proteins. Since histones are very basic proteins and have a higher isoelectric point (pH) compared to the medium, they become positively charged and migrate under an electric field. In the second dimension, histones are denatured by SDS and separated based on their molecular weight. The resulting 2D map can be used as a tool to enrich specific proteoforms at single-spot resolution for mass spectrometry analysis and immunological studies28,29. After electrophoresis, 2D TAU spots can be destained, trypsin-digested, and analyzed by mass spectrometry. Alternatively, the 2D TAU map can be blotted onto a nitrocellulose/PVDF filter and investigated by immunostaining27.
In this context, the methods might be strategic to improve the identification and analysis of novel post-translational modifications correlated with metabolic rewiring30,31.
We have further improved the original method by incorporating a sonication step, which is helpful for DNA degradation and improving protein solubility. Additionally, we have prolonged the incubation step for histone extraction, in order to obtain a protein sample with high purity. Moreover, in our protocol, we ran the first dimension at low voltage for a longer duration to achieve a better resolution. The resulting gel was used for investigating canonical histones PTMs and characterizing novel modifications such as glycation10,32.
Overall, we have developed a method for extracting and purifying histones, followed by the resolution of histone proteoforms using a two-dimensional map. The work also includes a procedure for in-gel digestion for mass spectrometry analysis and the steps for two-dimensional western blot analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>1,4-Dithioerythritol</td>
			<td>SIGMA- ALDRICH</td>
			<td>D8255&#160;</td>
			<td>Reagent for maintaining &#8722;SH groups in the reduced state; quantitatively reduces disulfides.</td>
		</tr>
		<tr>
			<td>1,5 M Tris-HCL buffer, pH 8.8</td>
			<td>BIO-RAD</td>
			<td>161-0798</td>
			<td>Resolving Gel Buffer</td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS&#160;</td>
			<td>BIO-RAD&#160;</td>
			<td>1610772</td>
			<td>Use this premixed 10x Tris/glycine/SDS running buffer to separate protein samples by SDS-PAGE.</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>33539</td>
			<td>2-Propanol (Isopropanol) is a secondary alcohol. It has been tested as a substitute to fuel for use in various fuel cells. CuO powder dissolved in 2-propanol has been used for the laser ablation assisted synthesis of Cu colloids. Suspension of 2-propanol with Zn(NO3)2 has been employed for the fabrication of titanium dioxide-coated stainless steel (P25-TiO2/SS) photoanode coated with uniformly thick layer of P25-TiO2.This photoanode was used for the electrochemical photocatalytic (ECPC) degradation process.</td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis Solution&#160;</td>
			<td>BIO-RAD</td>
			<td>1610158</td>
			<td>2.6% Crosslinker. Electrophoresis purity reagent&#160;</td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Carlo Erba Reagennts</td>
			<td>401392</td>
			<td>Acetic acid is an important chemical reagent with many industrial applicatios.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Panreac Applichem</td>
			<td>211007.1214</td>
			<td>Acetone&#160;is a solvent, renowned for its versatility in various laboratory, manufacturing, and cleaning applications.</td>
		</tr>
		<tr>
			<td>Acetonitile</td>
			<td>VWR</td>
			<td>83640320</td>
			<td>for HPLC&#160;&#160; LC-MS grade</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td>Agarose&#160;is a&#160;heteropolysaccharide&#160; frequently used in molecular biology.</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>SIGMA-ALDRICH</td>
			<td>A6141</td>
			<td>minimum 99,0%</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>A3678</td>
			<td>For molecular Biology, For electrophoresis, &#8805;98%&#160;</td>
		</tr>
		<tr>
			<td>Bioruptor plus</td>
			<td>Diagenode</td>
			<td>B01020001 - B01020002- B01020003</td>
			<td>sonicator</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>SIGMA- ALDRICH</td>
			<td>D9163-5G</td>
			<td>Reducing agent used to reduce disulfide bonds in proteins.</td>
		</tr>
		<tr>
			<td>Dodeca Silver Stain Kit&#160;</td>
			<td>BIO-RAD</td>
			<td>161-0480</td>
			<td>The Dodeca silver stain kit is easy-to-use for the detection of nonagram levels of proteins in polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>GE - Healthcare Life Sciences</td>
			<td>171325010L</td>
			<td>Glycerol is a simple triol compound, it is involved to aid in casting gradient gels, protein stabilizer and storage buffer component.</td>
		</tr>
		<tr>
			<td>Halt Phosphatase Inhibitor&#160;</td>
			<td>Thermo SCIENTIFIC</td>
			<td>78428</td>
			<td>Thermo Scientific Halt Phosphatase Inhibitor Cocktail preserves the phosphorylation state of proteins during and after cell lysis or tissue protein extraction&#160; Single- Use Cocktail (100X).&#160;&#160;</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor&#160;</td>
			<td>Thermo SCIENTIFIC</td>
			<td>78430</td>
			<td>Thermo Scientific Halt Protease Inhibitor Cocktail (100X) are ready-to-use concentrated stock solutions of protease inhibitors for addition to samples to prevent proteolytic degradation during cell lysis and protein extraction. Single- Use Cocktail (100X).&#160;&#160;</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>H1758</td>
			<td>BioReagent, for molecular biology</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>SIGMA-ALDRICH</td>
			<td>I6125</td>
			<td>&#8805;99% (NMR), crystalline</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SIGMA-ALDRICH</td>
			<td>P9541</td>
			<td>Potassium chloride, KCl, is&#160;generally used in laboratory routines. Its use as a storage buffer for pH electrodes and as a reference solution for conductivity measurements is well established.</td>
		</tr>
		<tr>
			<td>MCF10 cell line</td>
			<td>atcc</td>
			<td>CRL-10317&#160;</td>
			<td>epithelial cell line that was isolated in 1984 from the mammary gland of a White, 36-year-old female with fibrocystic breasts. This cell line was deposited by the Michigan Cancer Foundation.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>SIGMA-ALDRICH</td>
			<td>208337</td>
			<td>Magnesium chloride is a colorless crystalline solid.</td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylene-diamine</td>
			<td>SIGMA-ALDRICH</td>
			<td>T9281</td>
			<td>For Electrophoresis, approx. 99%</td>
		</tr>
		<tr>
			<td>Phosphate saline buffer 1X</td>
			<td>Corning</td>
			<td>15373631</td>
			<td>1 X PBS (phosphate buffered saline) is&#160;a buffered&#160;balanced salt solution&#160;used for a variety biological and cell culture applications, such as washing cells before dissociation, transporting cells or tissue, diluting cells for counting, and preparing reagents.</td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>60299</td>
			<td>Electron acceptor, employed in systems involving electron transport,</td>
		</tr>
		<tr>
			<td>SDS Solution 20% (w/v)&#160;</td>
			<td>BIO-RAD</td>
			<td>161-0418</td>
			<td>Sodium dodecyl sulfate. Electrophoresis purity reagent&#160;</td>
		</tr>
		<tr>
			<td>Sodium thiosulfate, ReagentPlus 99%</td>
			<td>SIGMA- ALDRICH</td>
			<td>21,726-3</td>
			<td>Sodium thiosulfate, ReagentPlus 99%</td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>339741</td>
			<td>Sulfuric Acid, Reagent, is&#160;an extremely corrosive acid that comes as yellowy slightly viscous liquid. It is soluble in water and is a diprotic acid. It has very strong corrosive, dehydrating and oxidizing properties as well as being hygroscopic.</td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>T6399-5G</td>
			<td>Trichloroacetic acid (TCA) is&#160;used as a reagent for the precipitation of proteins1,2 and nucleic acids3.&#160;</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>34957</td>
			<td>Trifluoroacetic acid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SIGMA- ALDRICH</td>
			<td>T9284-1L</td>
			<td>Triton X-100 is a nonionic polyoxyethylene surfactant that is most frequently used to extract and solubilize proteins.</td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>SIGMA-ALDRICH</td>
			<td>T6567</td>
			<td>Proteomics Grade, BioReagent, Dimethylated</td>
		</tr>
		<tr>
			<td>UREA</td>
			<td>SIGMA-ALDRICH</td>
			<td>33247</td>
			<td>Urea is a chaotropic agent and is used for protein denaturation. It disturbs the hydrogen bonds in the secondary, tertiary and quaternary structure of proteins. It can also disturb hydrogen bonding present in DNA secondary structure</td>
		</tr>
	</tbody>
</table>

unveiling histone proteoforms using 2d-tau gel electrophoresis

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.

In Figure 1A, it is possible to observe how histone proteins migrate in a 1D gel. This is an important preliminary step to understand the running time of the first dimension. Once the right time of separation is determined, run the second dimension. The 2D map of histone proteoforms shows a distinctive topographic pattern (Figure 1B). Each spot in Figure 1B represents a specific histone type. Observe the exact region of the gel wher...

Author Spotlight: Epigenetic Modifications and Metabolic Rewiring as Targets for Cancer Therapy

This method describes a simple and fast strategy for histone proteoform characterization and investigation.

Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which ...

unveiling-histone-proteoforms-using-2d-tau-gel-electrophoresis

Research

JoVE Journal

Biochemistry

387 Views.  Magna Græcia University of Catanzaro. This method describes a simple and fast strategy for histone proteoform characterization and investigation. 

Video: Author Spotlight: Epigenetic Modifications and Metabolic Rewiring as Targets for Cancer Therapy

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...