Quantitative Analysis of Posttranslational Modifications of Plant Histones

Histone Chaperone Deficiency in Arabidopsis Plants Triggers Adaptive Epigenetic Changes in Histone Variants and Modifications

“…In-depth investigations of plant epigenetics in histone chaperone or chromatin remodeling mutants are rare (this work and ( 35 )). Despite the substantial development of proteomic strategies, there is no universal approach for PTM characterization of all core histones and their variants, and the analytical setup has to be tailored to the main experimental objectives ( 45 , 56 ). To investigate histone epigenetics during plant chromatin remodeling while reducing bias in the quantification of histone forms, we applied an MS-based approach, including chemical derivatization of amine groups with propionic anhydride ( 44 ).…”

Unraveling Epigenetic Changes in A. thaliana Calli: Impact of HDAC Inhibitors

“…Histone extraction and derivatization were performed as described previously [ 23 , 40 , 41 ]. Nuclei in wash buffer (75 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl pH 8.0) were centrifuged at 3000× g and 4 °C for 10 min, incubated in nuclei lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM EDTA, 1% CHAPS) supplemented with protease, deacetylase, and phosphatase inhibitors (0.1 mM PMSF, 45 mM sodium butyrate, and 10 µL/mL protease inhibitor cocktail (P9599, Sigma-Aldrich) and 10 µL/mL phosphatase inhibitor cocktail (P5726, Sigma-Aldrich)) for 1 h on ice, spun at 10,000× g for 5 min, washed with 50 mM Tris-HCl, and spun at 10,000× g for 5 min.…”