Enhanced plant bottom-up histone proteomics

The available tools for characterization of histone proteoforms are essential for deciphering plant epigenetic mechanisms and their application in biotechnology. Insights into the epigenetic landscape of plant chromatin can be advanced using bottom-up proteomics. Mass spectrometry (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 Zea mays leaves on the quality of mass spectrometry data. We enhanced the MS-based plant histone analysis protocol by combining chemical derivatization using trimethylacetic anhydride with enzymatic digestion with a novel protease, Arg-C Ultra. Further, fluorescence-assisted cell sorting (FACS) has proven effective in isolating pure histone samples without the need for protein purification by precipitation. Proposed workflows produce highly pure histone extracts that are suitable for quantitative analysis of post-translational modifications and variant composition. They thus offer a powerful tool for investigating epigenetic patterns and their dynamics in agriculturally important crops.