The correct tools for characterization of histone proteoforms are essential for deciphering plant epigenetic mechanisms and their subsequent application in biotechnology. Insights into the epigenetic landscape of plant chromatin can be advanced using bottom-up proteomics. MS analysis of histone peptides relies on careful sample preparation, including chemical derivatization of amine groups prior to MS to improve their chromatographic behaviour during nanoHPLC separation. Characterizing histones in plant tissues remains especially challenging due to the presence of diverse, species-specific compounds that interfere with MS analysis. In this study, we evaluated the impact of different protocols for the preparation of histones from maize (Zea mays) leaves on the quality of MS data. We were able to enhance the MS-based plant histone analysis protocol by combining chemical derivatization using trimethylacetic anhydride with enzymatic digestion with a novel protease, Arg-C Ultra. In addition, fluorescence-assisted cell sorting proved to be effective in isolating pure histone samples without the need for protein purification by precipitation. Our proposed new workflows produce highly pure histone extracts that are suitable for quantitative analysis of post-translational modifications and variant composition. They therefore offer a powerful tool for investigating epigenetic patterns and their dynamics in agriculturally important crops.
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