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首页> 外文期刊>Journal of Experimental Botany >In vivo protein tyrosine nitration in Arabidopsis thaliana
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In vivo protein tyrosine nitration in Arabidopsis thaliana

机译：拟南芥体内蛋白质酪氨酸硝化

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摘要

Nitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, based on an anti-3-nitroY antibody, was performed to pull-down potential in vivo targets of Y nitration in the Arabidopsis thaliana proteome. Further shotgun liquid chromatography–mass spectrometry (LC-MS/MS) proteomic analysis of the immunoprecipitated proteins allowed the identification of 127 proteins. Around 35% of them corresponded to homologues of proteins that have been previously reported to be Y nitrated in other non-plant organisms. Some of the putative in vivo Y-nitrated proteins were further confirmed by western blot with specific antibodies. Furthermore, MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of protein spots, separated by two-dimensional electrophoresis from immunoprecipitated proteins, led to the identification of seven nitrated peptides corresponding to six different proteins. However, in vivo nitration sites among putative targets could not be identified by MS/MS. Nevertheless, an MS/MS spectrum with 3-aminoY318 instead of the expected 3-nitroY was found for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Reduction of nitroY to aminoY during MS-based proteomic analysis together with the in vivo low abundance of these modifications made the identification of nitration sites difficult. In turn, in vitro nitration of methionine synthase, which was also found in the shotgun proteomic screening, allowed unequivocal identification of a nitration site at Y287.
机译：蛋白质酪氨酸（Y）残基的硝化是一种翻译后修饰较低的修饰，可调节动物系统中蛋白质的功能或命运。但是，对于这种修饰及其在植物中的相应靶标的体内流行率知之甚少。进行了基于抗3-nitroY抗体的免疫沉淀，以降低拟南芥蛋白质组中Y硝化的体内潜在靶标。进一步的shot弹枪液相色谱-质谱（LC-MS / MS）蛋白质组学分析了免疫沉淀蛋白，从而鉴定出127种蛋白。其中约35％对应于先前已报道在其他非植物生物中被Y硝化的蛋白质的同源物。一些推定的体内Y-硝化的蛋白质通过用特异性抗体的蛋白质印迹进一步证实。此外，通过二维电泳从免疫沉淀的蛋白质中分离出的蛋白质斑点的MALDI-TOF（基质辅助激光解吸电离-飞行时间）分析可鉴定出对应于六个不同蛋白质的七个硝化肽。但是，MS / MS无法识别推定靶标中的体内硝化位点。然而，对于胞质甘油醛-3-磷酸脱氢酶，发现用3-氨基Y318代替预期的3-硝基Y的MS / MS谱图。在基于MS的蛋白质组学分析中，将硝基Y还原为氨基Y以及体内这些修饰的丰度较低，使得硝化位点的鉴定变得困难。反过来，在the弹枪蛋白质组学筛选中也发现了蛋氨酸合酶的体外硝化作用，可以明确鉴定Y287的硝化位点。

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