Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis
Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis
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基于二甲基标记的拟南芥定量乙酰蛋白质组学的建立
DOI：
10.1074/mcp.ra117.000530
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发表时间：
2018
影响因子：
7
通讯作者：
Li Ning
中科院分区：
文献类型：
--
作者：
Liu Shichang et al.;Li Ning
蛋白质乙酰化是多种翻译后修饰 (PTM) 类型之一，参与多种生物和细胞过程。在本研究中，我们应用基于 CsCl 密度梯度 (CDG) 离心的蛋白质分级和基于二甲基标记的 4C 定量 PTM 蛋白质组工作流程来研究拟南芥的动态乙酰蛋白质组变化。该工作流程将二甲基化学标记与基于色谱的乙酰肽分离和富集相结合，然后进行质谱 (MS) 分析、基于提取离子色谱 (XIC) 的质谱数据定量计算分析，使用名为稳定同位素定量二甲基标记 (SQUA-D) 的内部软件程序测量乙酰肽水平的动态变化，最后确认乙烯激素调节使用免疫印迹分析进行乙酰化。最终，利用这种蛋白质组学方法，从 2638 个不同的乙酰蛋白中发现了 7456 个明确的乙酰化位点，并重复鉴定了 5250 个乙酰化位点，其中包括赖氨酸侧链上的 5233 个位点和蛋白质 N 末端上的 17 个位点。在这些重复发现的乙酰化位点中，赖氨酸侧链上的 4228 个位点（即 80.5%）是新的。这些乙酰蛋白的例子有组蛋白超家族、核糖体和热休克蛋白以及与应激/刺激反应和能量代谢相关的蛋白质。 CDG 离心分离富集的新型乙酰蛋白含有许多细胞运输蛋白、膜结合受体和受体样激酶，它们主要参与油菜素类固醇、光、重力和发育信号传导。此外，我们还鉴定了组蛋白、P-糖蛋白、肌动蛋白解聚因子、ATP 酶、转录因子和受体样激酶内的 12 个高度保守的乙酰化位点基序。使用 SQUA-D 软件，我们定量了 33 个乙烯激素增强的乙酰肽组和 31 个激素抑制的乙酰肽组，或称为独特的 PTM 肽阵列 (UPA)，它们共享相同的独特 PTM 位点模式 (UPSP)。这种 CDG 离心蛋白质分级分离与基于二甲基标记的定量 PTM 蛋白质组学和 SQUA-D 相结合，可应用于任何模型真核生物和农作物以及动物和人类组织样本中任何 PTM 蛋白质的定量。
Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied bothCsCldensitygradient (CDG) centrifugation-based protein fractionation and a dimethyl-labeling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethylchemical labeling withchromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-basedcomputational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, namedStable isotope-basedQuantitation-Dimethyl labeling (SQUA-D), and finally theconfirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7456 unambiguous acetylation sites were found from 2638 different acetylproteins, and 5250 acetylation sites, including 5233 sites on lysine side chain and 17 sites on protein N termini, were identified repetitively. Out of these repetitively discovered acetylation sites, 4228 sites on lysine side chain (i.e.80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signaling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or calleduniquePTM peptidearrays (UPAs) that share the identicaluniquePTMsitepattern (UPSP). This CDG centrifugation protein fractionation in combination with dimethyl labeling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings.