In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana
In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana
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DOI：
10.1016/j.aca.2015.06.019
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发表时间：
2015-07-30
影响因子：
6.2
通讯作者：
Jin, Hong
中科院分区：
文献类型：
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作者：
Chen, Jiajia;Gao, Jun;Jin, Hong
组蛋白翻译后修饰 (PTM) 与染色质基因表达的遗传和表观遗传调控高度相关。质谱 (MS) 已发展成为鉴定和定量组蛋白 PTM 的最佳工具。使用丙酸酐、N-羟基琥珀酰亚胺酯（NHS-丙酸酯）等化学物质对组蛋白进行衍生化已广泛用于自下而上 MS 策略中的组蛋白 PTM 分析，这对组蛋白样品的纯度要求较高。然而，生物样品并不总是以高纯度制备，在大多数情况下含有去污剂或其他干扰物。作为一种替代方法，凝胶内衍生化方法（称为 In-gel NHS）在组蛋白 PTM 分析中得到了更广泛的应用，并且它被证明是一种更节省时间的制备方法。所提出的方法经过优化以获得更好的衍生化效率，并表现出较高的重现性，表明基于 In-gel NHS 的组蛋白 PTM 定量是可以实现的。在没有任何传统繁琐的组蛋白纯化程序的情况下，我们成功地利用 In-gel NHS 方法快速定量分析了拟南芥中选择性敲低 CLF (clf-29) 和原始亲本 (col) 的组蛋白 PTM，这表明 CLF 对拟南芥中的 H3K27me3 具有高度特异性。凝胶内 NHS 定量结果还表明植物中独特的组蛋白修饰模式，这为未来植物组蛋白修饰的研究奠定了宝贵的基础。 (C) 2015 Elsevier B.V. 保留所有权利。
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. (C) 2015 Elsevier B.V. All rights reserved.