Enhanced Plant Bottom-up Histone Proteomics

Join us to learn more about the Plant Bottom-up Histone Proteomics.

🔍 What you’ll learn:

Reliable characterization of histone proteoforms by bottom-up proteomics requires optimized sample preparation, as each processing step can substantially influence LC-MS/MS analytical performance, including peptide recovery, chromatographic behaviour, identification rates, and quantitative accuracy. This challenge is particularly pronounced in plant tissues, where abundant species-specific metabolites and other matrix components can interfere with histone isolation and downstream MS analysis.

In this study, the influence of different histone preparation workflows for maize (Zea mays) leaves on the quality of the resulting MS data was systematically evaluated. Particular attention was paid to key sample preparation steps, including histone isolation and purification, chemical derivatization, and proteolytic digestion.

Combining trimethylacetic anhydride derivatization with digestion by the novel protease Arg-C Ultra improved peptide generation and overall analytical performance compared to conventional approaches. Furthermore, fluorescence-assisted cell sorting enabled the isolation of highly pure histone samples while eliminating the need for precipitation-based purification steps, thereby reducing sample handling and potential losses. Peptide desalting using AttractSPE® Disks Tips C18 prior to LC-MS/MS analysis effectively removed residual salts, derivatization reagents, and other interfering compounds, resulting in improved chromatographic performance and enhanced signal quality. The optimized workflow produced cleaner extracts and improved the suitability of the samples for quantitative analysis of histone post-translational modifications and variant composition. These results demonstrate the critical impact of sample preparation strategy on the quality and interpretability of plant histone proteomics data and provide an improved workflow for epigenetic studies in agriculturally important crops.

Animated by Dr. Florine HALLEZ, Product Manager Bioanalysis, Affinisep

Guest speaker: Palina Ryzhaya, Central European Institute of Technology, Masaryk University (Czech Republic)

Biography: Palina is a 3rd-year PhD student at the Proteomics Core Facility, Mendel Centre for Plant Genomics and Proteomics (CEITEC). Her research focuses on MS-based histone proteomics and epigenetics in plant and mammalian systems. She is primarily interested in the development and optimization of sample preparation and LC-MS/MS workflows, and their further application in modern biological and biomedical research.

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