title: In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana.
abstract: Post-translational modifications (PTMs) on histone are 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 histone PTMs. Derivatization of histones with chemicals such as propionic anhydride, N-hydroxysuccinimide ester (NHS-propionate) has been widely used in histone PTMs analysis in bottom-up MS strategy, which requires high purity for histone samples. 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 derivatization method, termed In-gel NHS, is utilized for a broader application in histone PTMs analysis and it is shown to be a more time-saving preparation method. The proposed method was optimized for a better derivatization efficiency and displayed high reproducibility, indicating quantification of histone PTMs based on In-gel NHS was achievable. Without any traditional fussy histone purification procedures, we succeeded to quantitatively profile the histone PTMs 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 histone modification patterns in plants, which is invaluable foundation for future studies on histone modifications in plants.
authors: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors: Jiajia
authors: J
authors: Chen
authors: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China; China Novartis Institutes for BioMedical Research Co. Ltd., Building 8, Lane 898 Halei Road, Shanghai 201203, China.
authors: Jun
authors: J
authors: Gao
authors: State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors: Maolin
authors: M
authors: Peng
authors: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors: Yi
authors: Y
authors: Wang
authors: China Novartis Institutes for BioMedical Research Co. Ltd., Building 8, Lane 898 Halei Road, Shanghai 201203, China.
authors: Yanyan
authors: Y
authors: Yu
authors: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China. Electronic address: pyyang@fudan.edu.cn.
authors: Pengyuan
authors: P
authors: Yang
authors: Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China. Electronic address: jinhong@fudan.edu.cn.
authors: Hong
authors: H
authors: Jin
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D006657:Histones; D010940:Plant Proteins; D013388:Succinimides; C001426:N-hydroxysuccinimide
keywords: Epigenetic; Histone post-translational modifications; In-gel derivatization; Multiple reaction monitoring quantification