In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana.

Abstract Post-translational modifications (PTMs) on histoneare highly correlated with genetic and epigenetic regulation of gene expression from chromatin. Mass spectrometry (MS) has developed to be an optimal tool for the identification and quantification of histonePTMs. Derivatizationof histoneswith chemicals such as propionic anhydride, N-hydroxysuccinimideester (NHS-propionate) has been widely used in histonePTMs analysis in bottom-up MS strategy, which requires high purity for histonesamples. However, biological samples are not always prepared with high purity, containing detergents or other interferences in most cases. As an alternative approach, an adaptation of in gel derivatizationmethod, termed In-gel NHS , is utilized for a broader application in histonePTMs analysis and it is shown to be a more time-saving preparation method. The proposed method was optimized for a better derivatizationefficiency and displayed high reproducibility, indicating quantification of histonePTMs based on In-gel NHS was achievable. Without any traditional fussy histonepurification procedures, we succeeded to quantitatively profile the histonePTMs from Arabidopsis with selective knock down of CLF ( clf-29 ) and the original parental (col) with In-gel NHS method in a rapid way, which indicated the high specificity of CLF on H3K27me3 in Arabidopsis. In-gel NHS quantification results also suggest distinctive histonemodification patterns in plants, which is invaluable foundation for future studies on histonemodifications in plants.