Name: Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers (Video) | JoVE Journal
Uploaded: 2021-03-04T13:00:00.000+00:00
Duration: 7 min 10 s
Description: Learn to isolate high purity histones from sorghum leaf tissue for top-down mass spectrometry profiling of epigenetic markers. This method enables researchers to study drought resistance in crops.

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5 minutes

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.

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</ol>

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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>&lt;strong&gt;Disposables&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;ndash; 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><td></td></tr><tr><td>Water bath Sonicator</td><td>Fisher Scientific</td><td>15-336-120</td><td></td></tr></tbody></table>

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

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...

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. ...

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Learn to isolate high purity histones from sorghum leaf tissue for top-down mass spectrometry profiling of epigenetic markers. This method enables researchers to study drought resistance in crops.

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

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