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

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.

<ol>
	<li>Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+drought+stress:+Effects,+mechanisms+and+management.">Plant drought stress: Effects, mechanisms and management.</a> Agronomy for Sustainable Development. , 153-188 (2009).</li><li>Dai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drought+under+global+warming:+a+review.">Drought under global warming: a review.</a> Wiley Interdisciplinary Reviews: Climate Change. 2 (1), 45-65 (2011).</li><li>Rooney, W. L., Blumenthal, J., Bean, B., Mullet, J. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorghum+bicolor+-+an+important+species+for+comparative+grass+genomics+and+a+source+of+beneficial+genes+for+agriculture.">Sorghum bicolor - an important species for comparative grass genomics and a source of beneficial genes for agriculture.</a> Current Opinion in Plant Biology. 5 (2), 118-121 (2002).</li><li>Xu, 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=Drought+delays+development+of+the+sorghum+root+microbiome+and+enriches+for+monoderm+bacteria.">Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (18), 4284-4293 (2018).</li><li>Gao, C., 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=Strong+succession+in+arbuscular+mycorrhizal+fungal+communities.">Strong succession in arbuscular mycorrhizal fungal communities.</a> ISME Journal. 13 (1), 214-226 (2019).</li><li>Gao, C., 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=Fungal+community+assembly+in+drought-stressed+sorghum+shows+stochasticity,+selection,+and+universal+ecological+dynamics.">Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics.</a> Nature Communications. 11 (1), (2020).</li><li>Bannister, A. J., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Research. 21 (3), 381-395 (2011).</li><li>Yuan, L., Liu, X., Luo, M., Yang, S., Wu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+histone+modifications+in+plant+abiotic+stress+responses.">Involvement of histone modifications in plant abiotic stress responses.</a> Journal of Integrative Plant Biology. 55 (10), 892-901 (2013).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nature Reviews. Genetics. 10 (10), 669-680 (2009).</li><li>Huang, H., Lin, S., Garcia, B. A., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+analysis+of+histone+modifications.">Quantitative proteomic analysis of histone modifications.</a> Chemical Reviews. 115 (6), 2376-2418 (2015).</li><li>Moradian, A., Kalli, A., Sweredoski, M. J., Hess, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+top-down,+middle-down,+and+bottom-up+mass+spectrometry+approaches+for+characterization+of+histone+variants+and+their+post-translational+modifications.">The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications.</a> Proteomics. 14 (4-5), 489-497 (2014).</li><li>Sidoli, S., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+individual+histone+posttranslational+modifications+and+their+combinatorial+patterns+by+mass+spectrometry-based+proteomics+strategies.">Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies.</a> Methods in Molecular Biology. 1528, 121-148 (2017).</li><li>Maile, T. 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. V., 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=Identification+and+quantification+of+proteoforms+by+mass+spectrometry.">Identification and quantification of proteoforms by mass spectrometry.</a> Proteomics. 19 (10), 1800361 (2019).</li><li>Cupp-Sutton, K. 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. C., 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=A+cross-platform+toolkit+for+mass+spectrometry+and+proteomics.">A cross-platform toolkit for mass spectrometry and proteomics.</a> Nature Biotechnology. 30 (10), 918-920 (2012).</li><li>Kou, Q., Xun, L., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopPIC:+a+software+tool+for+top-down+mass+spectrometry-based+proteoform+identification+and+characterization.">TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization.</a> Bioinformatics (Ocford, England). 32 (22), (2016).</li><li>Park, J., 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=Informed-Proteomics:+open-source+software+package+for+top-down+proteomics.">Informed-Proteomics: open-source software package for top-down proteomics.</a> Nature Methods. 14 (9), 909-914 (2017).</li><li>LeDuc, R. D., 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+C-Score:+a+bayesian+framework+to+sharply+improve+proteoform+scoring+in+high-throughput+top+down+proteomics.">The C-Score: a bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics.</a> Journal of Proteome Research. 13 (7), 3231-3240 (2014).</li><li>Fornelli, 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=Advancing+top-down+analysis+of+the+human+proteome+using+a+benchtop+quadrupole-orbitrap+mass+spectrometer.">Advancing top-down analysis of the human proteome using a benchtop quadrupole-orbitrap mass spectrometer.</a> Journal of Proteome Research. 16 (2), 609-618 (2017).</li><li>Sun, R. 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=pTop+1.0:+A+high-accuracy+and+high-efficiency+search+engine+for+intact+protein+identification.">pTop 1.0: A high-accuracy and high-efficiency search engine for intact protein identification.</a> Analytical Chemistry. 88 (6), 3082-3090 (2016).</li><li>Xiao, K., Yu, F., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+protein+identification+using+isotopic+envelope+fingerprinting.">Top-down protein identification using isotopic envelope fingerprinting.</a> Journal of Proteomics. 152, 41-47 (2017).</li><li>Cai, W., 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=MASH+Suite+Pro:+A+comprehensive+software+tool+for+top-down+proteomics.">MASH Suite Pro: A comprehensive software tool for top-down proteomics.</a> Molecular &amp; Cellular Proteomics: MCP. 15 (2), 703-714 (2016).</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=Top-down+mass+spectrometry+of+histone+modifications+in+sorghum+reveals+potential+epigenetic+markers+for+drought+acclimation.">Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.</a> Methods. , (2019).</li><li>Garcia, B. A., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+combinatorial+modification+of+histone+H3+in+human+cells.">Pervasive combinatorial modification of histone H3 in human cells.</a> Nature Methods. 4 (6), 487-489 (2007).</li><li>Zheng, 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=Unabridged+analysis+of+human+histone+H3+by+differential+top-down+mass+spectrometry+reveals+hypermethylated+proteoforms+from+MMSET/NSD2+overexpression.">Unabridged analysis of human histone H3 by differential top-down mass spectrometry reveals hypermethylated proteoforms from MMSET/NSD2 overexpression.</a> Molecular &amp; Cellular Proteomics: MCP. 15 (3), 776-790 (2016).</li><li>Garcia, B. A., 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=Chemical+derivatization+of+histones+for+facilitated+analysis+by+mass+spectrometry.">Chemical derivatization of histones for facilitated analysis by mass spectrometry.</a> Nature Protocols. 2 (4), 933-938 (2007).</li><li>Holt, M. V., Wang, T., Young, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-pot+quantitative+top-+and+middle-down+analysis+of+GluC-digested+histone+H4.">One-pot quantitative top- and middle-down analysis of GluC-digested histone H4.</a> Journal of the American Society for Mass Spectrometry. 30 (12), 2514-2525 (2019).</li><li>Tian, Z., 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=Enhanced+top-down+characterization+of+histone+post-translational+modifications.">Enhanced top-down characterization of histone post-translational modifications.</a> Genome Biology. 13 (10), (2012).</li><li>Wang, Z., Ma, H., Smith, K., 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=Two-dimensional+separation+using+high-pH+and+low-pH+reversed+phase+liquid+chromatography+for+top-down+proteomics.">Two-dimensional separation using high-pH and low-pH reversed phase liquid chromatography for top-down proteomics.</a> International Journal of Mass Spectrometry. 427, 43-51 (2018).</li><li>Gargano, A. F. G., 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=Increasing+the+separation+capacity+of+intact+histone+proteoforms+chromatography+coupling+online+weak+cation+exchange-HILIC+to+reversed+phase+LC+UVPD-HRMS.">Increasing the separation capacity of intact histone proteoforms chromatography coupling online weak cation exchange-HILIC to reversed phase LC UVPD-HRMS.</a> Journal of Proteome Research. 17 (11), 3791-3800 (2018).</li></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 nuclei lysis step is critical; therefore, aggressive vortexing and pipetting should be avoided before adding NLB. In addition, loss of nuclei may occur when removing the supernatants from the pellets. Care must be taken to not disrupt the pellets when pipetting. The Triton X-100 concentration of 1% was optimized to selectively lyse the non-targeted organelles but not the nuclei (step 3.2). Optimal detergent concentration for other tissue or organisms may be different and need to be experimentally determined. Color change of the supernatant during the filtration process could indicate potential issues such as inefficient release of chloroplast or insufficient grinding of leaf. If possible, use a microscope to check for lysis of chloroplasts and retention of intact nuclei after each step to further optimize the protocol (especially if modifying the protocol for other tissues or plants). This protocol has only been tested with sorghum leaf tissue. It does not work for sorghum root tissue likely due to interference from soil. Application to other plant leaf tissues has not been tested and application to different plants may need additional optimization. For adapting the nuclei isolation protocol for ChIP-seq applications, an additional sucrose gradient density separation after step 3.3.4 (before using NLB) is advised to reduce cytoplasmic contamination. Because of the extensive clean-up steps, small amounts of residual non-nuclei materials are not expected to cause significant interference for histone analysis in LC-MS and can be left with the pellet.
Several initial trials failed when using commercial tablets of phosphatase inhibitors (e.g., PhosSTOP). The supernatant in step 3.1.6 appeared to be intense green when the tablets were used in the extraction buffer. The final extract showed low number of identified histones. We suspect the proprietary ingredients in the tablets may have caused nuclei lysis before step 3.4, reducing the overall histone yield. Another possible reason for failure is the incompatibility of the ingredients in the histone purification step with the ion exchange resin (step 3.4). We have used this protocol to consistently extract high purity histones for subsequent LC-MS over 150 samples. On average, we were able to obtain higher yield without using the &#8220;additional inhibitors&#8221; (unpublished data). Therefore, it is advised to cautiously test new inhibitors when modifying or adapting this protocol for other purposes. If phosphorylation is not of interest, the phosphatase inhibitors can be omitted in the extraction buffers.
The steps in 3.4 can take 3&#8211;4 h or more. It is recommended to break the protocol in 2 days&#8212;freeze the nuclei pellet from step 3.3 and perform the purification on day 2 (or later). The freeze-thaw cycle may partially help the nuclei lysis. The MWCO filter steps (3.4.7) can be very time consuming but can be easily scaled up by preparing multiple samples in parallel. Do not add the protease inhibitor tablets in step 3.4. Many commercial tablets contain polymers (e.g., polyethene glycol) as fillers, which will interfere with LC-MS analysis. At this step, the most other proteins should have been removed or denatured, so enzyme inhibitors are not critical. However, it is still necessary to keep the samples at 4 &#176;C or frozen to minimize degradation.
Following this protocol, histones can be successfully extracted from sorghum leaves. Histone PTMs can be characterized with LC-MS. The method can be potentially applied to large scale studies for comparing histone PTMs between different biological samples (e.g., different genotypes, plants grown under different conditions, etc.) as shown by the example data in Figure 3. However, data processing still requires extensive manual analysis for confidently assigning proteoforms, especially for unexpected (or novel) PTMs. New developments in bioinformatics tools are anticipated to automate the workflow and significantly increase the throughput for large-scale studies. Another limitation is that the top-down MS method, currently, cannot easily differentiate many proteoforms of hyper-modified H3 (e.g., multiple sites of mono/di/tri-metlylation and acetylation). The single dimension reversed-phase LC cannot fully separate the different H3 proteoforms. Therefore, the MS2 spectra of H3 will typically contain fragments from multiple proteoforms and cannot be easily and confidently deconvoluted. Combining top-down with bottom-up or middle-down methods30,32,33 can be especially beneficial for characterization of histone H3. Alternatively, multi-dimensional separation can be considered to improve the depth of top-down MS34,35,36.
Histone PTM profiling by LC-MS enables discovery of novel epigenetic markers for designing chromatin modifiers and improve the resilience of plants to severe environmental conditions. A pilot study using sorghum from two cultivars and grown under drought conditions in the field indicated that selective histone terminal clipping in leaf may be related to drought acclimation and plant development29. The identified histone markers may serve as targets by complementary techniques such as ChIP-seq. Comprehensive understanding of epigenetic factors gained from these complementary techniques would be indispensable for engineering innovative solutions to crops in response to environmental changes.

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

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 identified proteoforms, but do not overlap with the histone proteoforms eluting in the later stage of the LC gradient. The results can be easily visualized with the latest TopPIC or Informed-Proteomics packages. For demonstration, we will focus on the data visualization using the Informed-Proteomics package, which can be used to directly load raw MS files and manually examine proteoform identifications. Please note that both software packages use different algorithms and parameters. The reported numbers of proteoforms will not be identical. We recommend reporting the proteoform counts from TopPIC because it is more conservative, and it does consider unknown PTMs. Informed-Proteomics package has integrated data processing and visualization for easy manual validation. For organisms with well-annotated PTMs, we recommend ProSightPC24 for best site localization. Combining the results using multiple tools can increase the number of and the confidence of proteoform identifications.
After processing the data with Informed-Proteomics, the LC-MS feature map can be visualized in LcMsSpectator, which displays the deconvoluted protein masses against the LC retention time. By clicking on the identified proteoforms in the software, the associated feature will be highlighted with a small green rectangle in the feature map. Major histone proteins should be seen in specific regions of the map, which indicates the success of the experiment. Figure 1a shows a representative LC-MS feature map of intact histones. Full-length histone proteoforms are highlighted in the dashed boxes. Most proteoforms detected can be confidently identified using MS2 data.
Figure 1b shows the zoom in of the region with H2A and H2B proteoforms. Most of them have N-terminal modifications of 42 Da. This nominal mass corresponds to either trimethylation (42.05 Da) or acetylation (42.01 Da), which are commonly seen for histones. Their accurate masses differ only by 0.04 Da and are difficult to differentiate at the intact protein level (~2 ppm). In high resolution MS2 spectra, the PTMs can be easily differentiated and confirmed because of the lower mass of the fragments29. In addition, H2A and H2B histones have multiple homologs with very similar sequences as noted by the different UniProt accession numbers in Figure 1b. Again, high resolution LC-MS analysis can readily identify and differentiate them. Two types of H2As were identified for sorghum histones. The 16 kDa H2A histones in Figure 1b have extended terminal tails in the non-conserved regions of histones. Another group of H2A histones without the extended tails (14 kDa) can be seen in Figure 1c.
For H4 histones, N-terminal acetylation was identified as the major PTM. Additional lysine acetylations and methionine oxidations can be also observed simply by examining the mass differences of the features in Figure 1d. We also observed an unknown modification of 112.9 Da in addition to the N-terminal acetylation (the feature above &#8220;3Ac&#8221; in Figure 1d). This is likely some unknown adducts from the reagent used in the preparation. We have previously detected sulfate ion adducts on H4, which may be attributed to residual salts combined with high basicity of histone proteins. For H3, two protein sequences were identified H3.3 and H3.2 (Figure 1e). Although these two protein sequences differ at only 4 residues (32, 42, 88, and 91), they can still be easily distinguished in LC-MS based on the separation in both dimensions, mass, and retention time. H3 proteins are heavily modified by varying degrees of methylation and acetylation. The high degree of modification can be easily visualized by the dense, parallel lines in the feature map, which are 14 Da apart. However, three methylation groups (14*3 Da) have the equal nominal mass to one acetylation (42 Da). Because these PTMs cannot be easily resolved at intact protein level, they are referred to as &#8220;methyl equivalents&#8221; (i.e., multiples of 14 Da; one acetylation equals three methyl equivalents). In Figure 1e, H3 proteoforms are labeled in the form of methyl equivalents based on their intact mass. Due to limited resolution of the RPLC separation, many different H3 proteoforms are likely co-eluting and fragmented in the same spectrum. The method presented here will only identify the most abundant combinations of methylation and acetylation as illustrated in Figure 2. For more comprehensive characterization of H3, more targeted analysis is still required30,31.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61707/61707fig01.jpg" /> 
Figure 1: LC-MS feature map on intact histones extracted from sorghum leaves.&#160;The figure shows LC retention time (in minutes) vs. the molecular mass for all detected proteoforms. The log abundance is shown by the color scale next to the top map (log 10 abundance). (a) The major histone peaks are labeled by the dashed boxes. Most of the features outside the boxes are truncated histones and ribosomal proteins. Zoom-in views for each group of histones: (b) H2B and 16 kDa H2A, (c) H3, (d) 14 kDa H2A, and (e) H3. The UniProt accession numbers are noted alongside each feature, followed by detected PTMs. &#8220;Ac&#8221;, &#8220;me&#8221;, &#8220;+O&#8221; indicate acetylation, methylation, and oxidation, respectively. In (b), two truncated H2A C5YZA9 proteoforms are labeled, which had one or two C-terminal alanine clipped (shown as -A*, and -AA*). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61707/61707fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A representative example of proteoform identification is shown in Figure 2 using MSPathfinder and visualized in LcMsSpectator. The fragmentation spectrum in Figure 2a was generated using ETD, which yields c and z type ions along the protein backbone. HCD of the same precursor can be used to validate the identification, but HCD generally provides limited sequence coverage20. The precursor ions in the previous and next MS1 spectra are shown in Figure 2b,c, with their matched isotope peaks highlighted in purple. The sequence coverage map in Figure 2d can help localize any possible PTMs. A high-confidence identification should have most of the fragments matched, precursor ion matched, and good sequence coverage to help localize PTMs. In this example, an H3.2 proteoform was identified with two PTMs&#8212;di-methylation on K9 and methylation on K27. Following the same method, other proteoforms with different PTMs and terminal truncations can be manually validated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61707/61707fig02.jpg" /> 
Figure 2: Representative example of an identified histone H3.2 proteoform.&#160;H3.2 preteoform with its (a) ETD spectrum, (b) precursor ion in the previous MS1 spectrum, (c) precursor ion in the next MS1 spectrum, and (d) sequence coverage map. The c ions from the N-terminus are labeled in cyan, and the z ions from the C-terminus are in pink. Two PTMs were identified and highlighted in yellow in (d) with their mass shifts annotated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61707/61707fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Quantitative comparison of the detected histone proteoforms can reveal potential epigenetic markers. We have applied this protocol previously to 48 sorghum samples collected from the field (&#8220;additional inhibitors&#8221; were not used in this study)29. Two different genotypes of sorghum were compared in response to pre-flowering or post-flowering droughts. By comparing the relative abundance of the proteoforms, we discovered some interesting changes of truncated histone proteoforms that are specific to sample conditions as shown in Figure 3. C-terminal truncation of H4 was observed only in weeks 3 and 9 for some of the samples (Figure 3a,b). For H3.2, N-terminal truncated proteoforms were generally more abundant in week 10 (Figure 3c,d). In contrast, C-terminal truncated H3.2 tend to be seen in earlier time points (Figure 3c). More importantly, the two genotypes did not respond in the exact same way. The H4 C-terminal truncated proteoforms were significantly more abundant in BTx642 than in RTx430 (Figure 3b). Such data reveals potential epigenetic markers of plant development and stress tolerance that can be further tested with other techniques.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61707/61707fig03.jpg" /> 
Figure 3: Quantitative comparison of histone proteoforms.&#160;(a) Heatmap of histone H4 proteoforms across different samples. For each proteoform, the abundance extracted from top-down MS data was normalized to the sum of all identified H4 proteoforms in each analysis, yielding the &#8220;relative abundance&#8221;. The values were then scaled to the maximum of each row to better show the changes in low abundance proteoforms. The scaled relative abundance is denoted in the color key at the bottom of the heatmap. Growth conditions are noted on the horizontal axis (Pre: pre-flowering drought, Post: post-flowering drought). Three replicates are grouped together and are separated by black vertical stripes from other conditions. For samples labeled with asterisks, only technical replicates were acquired. Proteoforms are represented on the vertical axis, in the format &#8220;starting residue &#8211; ending residue: mass; putative modification&#8221;. (b) Relative abundance plot of the truncated H4 proteoforms 2&#8211;99 (proteoforms highlighted in bold in (a) are summed) at different conditions. The key to the symbols is shown in the legend in the top-right corner. Filled dots in the middle of the error bars are the average values. (c) Heatmap of H3.2 proteoforms and (d) abundance plot for all identified N-terminal truncated H3.2 are shown in the same format as those for H4. Proteoforms smaller than 8&#8239;kDa in (c) were omitted for simplicity. The N-terminal and C-terminal truncated H3.2 proteoforms showed different responses across the growth conditions. Reprinted with permission from ELSEVIER from ref.29. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61707/61707fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

isolation-histone-from-sorghum-leaf-tissue-for-top-down-mass

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.5K Views.  Pacific Northwest National Laboratory. Ons protocol onthult epigenetische markers voor de ontwikkeling van sorghumplanten en droogtebestendigheid. Een dergelijk moleculair begrip zal helpen betere oplossingen te ontwikkelen om gewassen in de toekomst aan te passen aan extreme klimaten. Dit protocol genereert hoge zuiverheid histonen uit sorghum bladweefsel geschikt voor niet-gerichte profilering van post-translationele modificaties door massaspectrometrie.Shadan Abdali en Tanya Winkler, post-bachelor onderzoeksmedewerkers v...

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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. ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

Genetics

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Chemistry

2D Gel Separation for Histone Isoform Enrichment and PTM Characterization

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.

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.

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

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Biology

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Neuroscience

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

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

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

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...

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

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

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

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

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