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

• DOI: 10.3791/61707
• Publication Date: 2021-03-05T00:00:16Z
• Authors: Mowei Zhou; Shadan H. Abdali; David Dilworth; Lifeng Liu; Benjamin Cole; Neha Malhan; Amir H. Ahkami; Tanya E. Winkler; Joy Hollingsworth; Julie Sievert; Jeff Dahlberg; Robert Hutmacher; Mary Madera; Judith A. Owiti; Kim K. Hixson; Peggy G. Lemaux; Christer Jansson; Ljiljana Paša-Tolić
• Keywords: 0301 basic medicine; 1.1 Normal biological development and functioning; Buffers; Mass Spectrometry; Epigenesis, Genetic; Histones; 03 medical and health sciences; Genetic; Underpinning research; Genetics; Psychology; Amino Acid Sequence; Protein Processing; Sorghum; Plant Proteins; 580; Cell Nucleus; 2. Zero hunger; Chromatography; Liquid; Post-Translational; Biological Sciences; Plant Leaves; Biochemistry and cell biology; Cognitive Sciences; Biochemistry and Cell Biology; Protein Processing, Post-Translational; Biomarkers; Epigenesis; Chromatography, Liquid

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.