We thank Ronald Moore and Thomas Fillmore for helping with mass spectrometry experiments, and Matthew Monroe for data deposition. This research was funded by grants from US Department of Energy (DOE) Biological and Environmental Research through the Epigenetic Control of Drought Response in Sorghum (EPICON) project under award number DE-SC0014081, from the US Department of Agriculture (USDA; CRIS 2030-21430-008-00D), and through the Joint BioEnergy Institute (JBEI), a facility sponsored by DOE (Contract DE-AC02-05CH11231) between Lawrence Berkeley National Laboratory and DOE. The research was performed using Environmental Molecular Sciences Laboratory (EMSL) (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research.

1. Preparing sorghum leaf material
NOTE: The sorghum plants were grown in soil in the field in Parlier, CA.
<ol>
	<li>Collect sorghum leaves from plants into 50 mL centrifuge tubes and immediately freeze the tube in liquid nitrogen. Collect leaf tissue by tearing off the third and fourth fully emerged leaf from the primary tiller. 
	NOTE: More details of field condition, sample growth, and collection can be found in the published report18.</li>
	<li>Grind leaves ...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+sorghum+as+a+dedicated+bioenergy+feedstock.">Designing sorghum as a dedicated bioenergy feedstock.</a> Biofuels, Bioproducts and Biorefining. 1 (2), 147-157 (2007).</li><li>Mullet, J. E., Klein, R. R., Klein, P. 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M., 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=Mass+spectrometric+quantification+of+histone+post-translational+modifications+by+a+hybrid+chemical+labeling+method.">Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.</a> Molecular &amp; Cellular Proteomics. 14 (4), 1148-1158 (2015).</li><li>Dang, X., 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+first+pilot+project+of+the+consortium+for+top-down+proteomics:+a+status+report.">The first pilot project of the consortium for top-down proteomics: a status report.</a> Proteomics. 14 (10), 1130-1140 (2014).</li><li>Schaffer, L. 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A., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+quantitative+top-down+proteomics.">High-throughput quantitative top-down proteomics.</a> Molecular Omics. , (2020).</li><li>Varoquaux, N., 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=Transcriptomic+analysis+of+field-droughted+sorghum+from+seedling+to+maturity+reveals+biotic+and+metabolic+responses.">Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (52), 27124 (2019).</li><li>Gordon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vanadate+as+protein-phosphotyrosine+phosphatase+inhibitor.">Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.</a> Methods in Enzymology. 201, 477-482 (1991).</li><li>Zhou, M., 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=Profiling+changes+in+histone+post-translational+modifications+by+top-down+mass+spectrometry.">Profiling changes in histone post-translational modifications by top-down mass spectrometry.</a> Methods in Molecular Biology. 1507, 153-168 (2017).</li><li>Chambers, M. 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The presented protocol describes how to extract histones from sorghum leaf (or more generally plant leaf) samples. The average histone yield is expected to be 2&#8211;20 &#181;g per 4&#8211;5 g sorghum leaf material. The materials are sufficiently pure for the downstream histone analysis by LC-MS (mostly histones with ~20% ribosomal protein contamination). Lower yield may be obtained due to sample variations, or potential mishandling/failures throughout the protocol. Maintaining the integrity of the nuclei before the nuc...

The increasing severity and frequency of drought is expected to affect productivity of cereal crops1,2. Sorghum is a cereal food and energy crop known for its exceptional ability to withstand water-limiting conditions3,4. We are pursuing mechanistic understanding of the interplay between drought stress, plant development, and epigenetics of sorghum [Sorghum bicolor (L.) Moench] plants. Our previous work has demonstrated strong connections between plant and rhizosphere microbiome in drought acclimation and responses at the molecular level5,6,7. This research will pave the way for utilizing epigenetic engineering in adapting crops to future climate scenarios. As a part of the efforts in understanding epigenetics, we aim to study protein markers that impact gene expression within the plant organism.
Histones belong to a highly conserved family of proteins in eukaryotes that pack DNA into nucleosomes as fundamental units of chromatin. Post-translational modifications (PTMs) of histones are dynamically regulated to control chromatin structure and influence gene expression. Like other epigenetic factors, including DNA methylation, histone PTMs play important roles in many biological processes8,9. Antibody-based assays such as western blots have widely been used to identify and quantify histone PTMs. In addition, the interaction of histone PTMs and DNA can be effectively probed by Chromatin immunoprecipitation &#8211; sequencing (ChIP-seq)10. In ChIP-seq, chromatin with specific targeted histone PTM is enriched by antibodies against that specific PTM. Then, the DNA fragments can be released from the enriched chromatin and sequenced. Regions of genes that interact with the targeted histone PTM are revealed. However, all these experiments heavily rely on high quality antibodies. For some histone variants/homologs or combinations of PTMs, development of robust antibodies can be extremely challenging (especially for multiple PTMs). In addition, antibodies can only be developed if the targeted histone PTM is known.11 Therefore, alternative methods for untargeted, global profiling of histone PTMs are necessary.
Mass spectrometry (MS) is a complementary method to characterize histone PTMs, including unknown PTMs for which antibodies are not available11,12. The well-established &#8220;bottom-up&#8221; MS workflow uses proteases to digest proteins into small peptides prior to liquid chromatography (LC) separation and MS detection. Because histones have large numbers of basic residues (lysine and arginine), the trypsin digestion (protease specific to lysine and arginine) in the standard bottom-up workflow cuts the proteins into very short peptides. The short peptides are technically difficult to analyze by standard LC-MS, and do not preserve the information about the connectivity and stoichiometry of multiple PTMs. The use of other enzymes or chemical labeling to block lysines generates longer peptides that are more suitable for characterization of histone PTMs13,14.
Alternatively, the digestion step can be completely omitted. In this &#8220;top-down&#34; approach, intact protein ions are introduced into the MS by electrospray ionization (ESI) after online LC separation, yielding ions of the intact histone proteoforms. In addition, ions (i.e., proteoforms) of interest can be isolated and fragmented in the mass spectrometer to yield the sequence ions for identification and PTM localization. Hence, top-down MS has the advantage to preserve the proteoform-level information and capture the connectivity of multiple PTMs and terminal truncations on the same proteoform15,16. Top-down experiments can also provide quantitative information and offer insights of biomarkers at the intact protein level17. Herein, we describe a protocol to extract histone from sorghum leaf and analyze the intact histones by top-down LC-MS.
The example data shown in Figure 1 and Figure 2 are from sorghum leaf collected at week 2 after planting. Although variation of yield is expected, this protocol is generally agnostic to specific sample conditions. The same protocol has been successfully used for sorghum plant leaf tissue collected from 2, 3, 5, 8, 9, and 10 weeks after planting.

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>43815-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 500mM Solution, pH 8.0</td>
			<td>EMD Millipore Corp</td>
			<td>324504-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine Hydrochloride</td>
			<td>Sigma</td>
			<td>G3272-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic</td>
			<td>Sigma</td>
			<td>P3786-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, cOmplete tablets</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Sigma</td>
			<td>303410-5G</td>
			<td>Used for histone deacetylase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S7020-100G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>450243-10G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Weak cation exchange resin, mesh 100-200 analytical (BioRex70)</td>
			<td>Bio-Rad</td>
			<td>142-5842</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography column (Bio-Spin)</td>
			<td>BIO-RAD</td>
			<td>732-6008</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh 100 filter cloth</td>
			<td>Millipore Sigma</td>
			<td>NY1H09000</td>
			<td>This is part of the Sigma kit (catalog # CELLYTPN1) for plant nuclei extraction. Similar filters with the same mesh size can be used.</td>
		</tr>
		<tr>
			<td>Micropipette tips (P20, P200, P1000)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, 50mL/15mL, Centrifuge, Conical</td>
			<td>Genesee Scientific</td>
			<td>28-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Microcentrifuge, 1.5/2 mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Fisher Scientific</td>
			<td>01-912-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers (50mL &#8211; 2L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td>13-690-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging-bucket centrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>50-728-002</td>
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		</tr>
		<tr>
			<td>Water bath Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15-336-120</td>
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</table>

isolation of histone from sorghum leaf tissue for top down mass spectrometry profiling of potential epigenetic markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

Following the protocol, the histones can be extracted and identified using the LC-MS analysis. The raw data and processed results are available at MassIVE (https://massive.ucsd.edu/) via accession: MSV000085770. Based on the TopPIC results from the representative sample (available also from MassIVE), we identified 303 histone proteoforms (106 H2A, 72 H2B, 103 H3, and 22 H4 proteoforms). Co-purified ribosomal proteoforms have also been detected, typically eluting early in the LC. They usually consist of ~20% of the identi...

Watch this Scientific Journal Video about Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers at JoVE.com

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Our protocol uncovers epigenetic markers for sorghum plant development and drought resistance. Such molecular understanding will help develop better solutions to adapt crops for extreme climates in the future. This protocol generates high purity histones from sorghum leaf tissue suitable for un-targeted profiling of post-translational modifications by mass spectrometry.Demonstrating the procedure will be Shadan Abdali and Tanya Winkler, post-bachelor research associates from EMSL. To begin, grind a few sorghum leaves with liquid nitrogen and store them in a 50 milliliter centrifuge tube at minus 80 degrees Celsius. Use approximately four grams of the leaf powder for histone analysis of each sample.Prepare extraction buffers 1, 2A, and 2B as described in the text manuscript and add a protease inhibitor tablet to extraction buffer one to make a final concentration of 0.2X. Then add 20 milliliters of the prepared extraction buffer one to the ground leaf powder and gently mix or vortex for 10 minutes. Using mesh 100, rinse the filtered material twice with two milliliters of extraction buffer one each time.Next, centrifuge the filtrate at 3, 000 times G for 10 minutes at four degrees Celsius in a swinging bucket rotor to pellet the cell debris and large subcellular organelles. Simultaneously, prepare extraction buffer 2A by adding protease inhibitor to a final concentration of 0.4X. Discard the supernatant without disturbing the pellet.Then resuspend the pellet in five milliliters of the prepared extraction buffer 2A. Incubate it on ice for 10 minutes with gentle mixing. Centrifuge the solution at 2, 100 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor to pellet the debris and nuclei.Decant the supernatant carefully without disturbing the pellet. Prepare extraction buffer 2B by adding the protease inhibitors to a final concentration of 1X. Add five milliliters of this prepared buffer to the pellet obtained after the centrifugation.Centrifuge at 2, 100 times G for 15 minutes at four degrees Celsius in the swinging bucket rotor to pellet debris and nuclei. Simultaneously, prepare nuclei lysis buffer by adding a protease inhibitor tablet. Carefully decant the supernatant without disturbing the pellet and resuspend the pellet in 250 microliters of the lysis buffer.Vortex the solution for 15 seconds at maximum speed to homogenize it and resuspend the material. Then sonicate it for five minutes at four degrees Celsius and store it at minus 80 degrees Celsius. Thaw the frozen nuclei sample and add 750 microliters of 5%guanidine buffer.Then sonicate it for 15 minutes at four degrees Celsius. Transfer the sample to a two milliliter tube and spin it at 10, 000 times G for 10 minutes at four degrees Celsius. Simultaneously, prepare an ion exchange chromatography column by rinsing it with two milliliters of acetonitrile and four milliliters of water to minimize contamination on the surface.Load approximately 200 to 300 microliters of weak cation exchange resin onto the chromatography column and let the resin settle. Wash the resin four times with guanidine buffer and place the tube and column on ice. Put the column on a two milliliter collection tube and load the supernatant from the prepared sample slowly onto the resin bed without disrupting the resin.As the solution is flowing through, load the eluent back to the top of the column six to eight times to allow maximum binding to the resin. Then load two milliliters of 5%guanidine buffer to wash the non-histone proteins off the column. Elute the histone proteins with one milliliter of 5%guanidine buffer and collect the eluent.Clean a three kilodalton cutoff spin filter with 500 microliters of wash solvent, then desalt the remaining eluent using the pre-cleaned spin filter. Major histone proteins were observed in specific regions of the LC-MS feature map indicating the success of the experiment. This representative map shows intact full-length histones in dashed boxes.The LC-MS feature map of H2B and 16 kilodalton H2A proteoforms shows that both have multiple homologs with similar sequences as noted by the unique product session numbers. Another group of H2A histones without the extended tails can be seen. The N-terminal acetylation, additional lysine acetylations, and methionine oxidations in H4 histone proteins can be observed by examining the mass differences in the LC-MS feature map shown here.There were two protein sequences identified for H3 histone proteins, H3.3 and H3.2. Electron transfer dissociation fragmentation was used to generate the fragmentation spectrum of the identified H3.2 proteoform. The precursor ions and the previous and next Ms1 spectra are shown here with their matched isotope peaks highlighted in purple.Post-translational modifications can be localized using the sequence coverage map. By comparing the relative abundance of the proteoforms, changes of truncated histone proteoforms specific to sample conditions were discovered. C-terminal truncation of H4 was observed only in weeks three and nine for some of the samples.For H3.2, N-terminal truncated proteoforms were generally more abundant in week 10. In contrast, C-terminal truncated H3.2 were seen in earlier time points. The H4 C-terminal truncated proteoforms were significantly more abundant in BTx642 than in RTx430.When attempting this protocol, pay close attention to the colors of the supernatant and the pellet. The color change help identify potential problems in the grinding or lysis of the chloroplast. We hope that this robust protocol for histone isolation from sorghum leaves will enable epigenetic research for other similar plants.

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Biochemistry

4.5K Views.  Pacific Northwest National Laboratory. Il nostro protocollo scopre marcatori epigenetici per lo sviluppo di piante di sorgo e la resistenza alla siccità. Tale comprensione molecolare aiuterà a sviluppare soluzioni migliori per adattare le colture ai climi estremi in futuro. Questo protocollo genera istoni ad alta purezza dal tessuto fogliare di sorgo adatto per la profilazione non mirata di modifiche post-tras tras trasutzionali mediante spettrometria di massa.A dimostrare la procedura saranno Shadan Abdali e Tanya Winkle...