Identification of post-translational modifications of plant protein complexes.
Identification of post-translational modifications of plant protein complexes.
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DOI：
10.3791/51095
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发表时间：
2014-02-22
期刊：
影响因子：
--
通讯作者：
Ntoukakis V
中科院分区：
文献类型：
--
作者：
Piquerez SJ;Balmuth AL;Sklenář J;Jones AM;Rathjen JP;Ntoukakis V
由于复杂的感知和信号系统，植物能够迅速适应不断变化的环境。在病原体侵袭期间，植物通过免疫复合物的募集和激活对感染做出快速反应。免疫复合物的激活与蛋白质的翻译后修饰（PTMs）有关，如磷酸化、糖基化或泛素化。了解这些ptm是如何编排的，将有助于更好地理解如何实现抵抗。在这里，我们描述了一种核苷酸结合富亮氨酸重复（NB-LRR）相互作用蛋白的蛋白质纯化方法，并随后鉴定了它们的翻译后修饰（PTMs）。经过少量修改，该方案可用于其他植物蛋白复合物的纯化。该方法基于感兴趣蛋白的表位标记版本的表达，随后通过免疫沉淀部分纯化，并进行质谱分析以鉴定相互作用蛋白和PTMs。该协议表明：i)。质谱法可以检测PTMs的动态变化，如磷酸化；(二)。重要的是要有足够数量的目标蛋白，这可以弥补免疫沉淀纯度的不足；iii)。为了检测感兴趣的蛋白质的ptm，必须对该蛋白质进行免疫沉淀以获得足够数量的蛋白质。
Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
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影响因子：
5.6
作者：
Bonardi V;Dangl JL
通讯作者：
Dangl JL
影响因子：
11.6
作者：
Ishihama, Nobuaki;Yamada, Reiko;Yoshioka, Hirofumi
通讯作者：
Yoshioka, Hirofumi
DOI：
10.1073/pnas.0909705107
发表时间：
2010-01-05
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
11.4
作者：
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通讯作者：
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影响因子：
30.3
作者：
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通讯作者：
Coaker G