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

Abstract

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.

Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana

“…Protocols using these core methods have been shown to produce high‐purity histone extractions and high‐quality MS data in several model organisms, including humans, mice, flies, and worms (Garcia et al., 2007; Haws et al., 2020; Holt et al., 2021; L. Johnson, 2004; Jung et al., 2010; Karch et al., 2013; Shechter et al., 2007; Sidoli, Simithy, Karch, Kulej, & Garcia, 2015; Zhou et al., 2017). However, most protocols for histone extraction have been designed for non‐plant organisms and produce low‐purity histones or low‐quality data when used in plants, evidenced by the requirement for additional purification steps, such as gel excision and chromatography, or the use of membranes to remove non‐histone proteins and other impurities in existing plant‐based methods (Chen et al., 2015; L. Johnson, 2004; Ledvinová et al., 2018; Moraes et al., 2015; Zhou et al., 2021). Additional purification steps complicate the overall workflow and extend the required time to purify histones when compared to protocols used in other organisms.…”

“…Correspondingly, this could lead to insufficient sample content for alternative assays for verification purposes and further analysis. Furthermore, current plant‐based histone purification methods used for MS require additional purification steps, including gel excision, chromatography, or the use of membranes, to effectively remove non‐histone proteins and other impurities, which complicate the overall workflow (Chen et al., 2015; Johnson, 2004; Ledvinová et al., 2018; Moraes et al., 2015; Zhou et al., 2021). Therefore, a simple protocol centered on plant‐based histone extractions that would allow for both purity and high yield is greatly needed.…”