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><tbody><tr><td>1,4-Dithioerythritol</td><td>SIGMA- ALDRICH</td><td>D8255&amp;nbsp;</td><td>Reagent for maintaining &amp;minus;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&amp;nbsp;</td><td>BIO-RAD&amp;nbsp;</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&amp;nbsp;</td><td>BIO-RAD</td><td>1610158</td><td>2.6% Crosslinker. Electrophoresis purity reagent&amp;nbsp;</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&amp;nbsp;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&amp;nbsp;&amp;nbsp; LC-MS grade</td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>15510-027</td><td>Agarose&amp;nbsp;is a&amp;nbsp;heteropolysaccharide&amp;nbsp; 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, &amp;ge;98%&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp; Single- Use Cocktail (100X).&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Halt Protease Inhibitor&amp;nbsp;</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).&amp;nbsp;&amp;nbsp;</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>&amp;ge;99% (NMR), crystalline</td></tr><tr><td>KCl</td><td>SIGMA-ALDRICH</td><td>P9541</td><td>Potassium chloride, KCl, is&amp;nbsp;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&amp;nbsp;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>208337</td><td>Magnesium chloride is a colorless crystalline solid.</td></tr><tr><td>N,N,N&#039;,N&#039;-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&amp;nbsp;a buffered&amp;nbsp;balanced salt solution&amp;nbsp;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)&amp;nbsp;</td><td>BIO-RAD</td><td>161-0418</td><td>Sodium dodecyl sulfate. Electrophoresis purity reagent&amp;nbsp;</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&amp;nbsp;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&amp;nbsp;used as a reagent for the precipitation of proteins&lt;sup&gt;1,2&lt;/sup&gt; and nucleic acids&lt;sup&gt;3&lt;/sup&gt;.&amp;nbsp;</td></tr><tr><td>Trifluoroacetic acid</td><td>Riedel-de Ha&amp;euml;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

410 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

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Cancer Research

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Genetics

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Source: Danis, C. et. al., <a href="https://app.jove.com/v/55001">Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.