Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE):: LC-ESI-MS/MS evidence for Cys374 Michael adduction
Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE):: LC-ESI-MS/MS evidence for Cys374 Michael adduction
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DOI：
10.1002/jms.872
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发表时间：
2005-07-01
影响因子：
2.3
通讯作者：
Carini, M
中科院分区：
文献类型：
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作者：
Aldini, G;Dalle-Donne, I;Carini, M
我们首次通过质谱学和计算相结合的方法证明了G-和F-肌动蛋白可以被脂类衍生的醛，4-羟基-反式-2-壬烯醛共价修饰，提供了修饰蛋白质的分子质量以及加合机理和位置的信息。不同摩尔比的HNE(1：1到1：20)处理的肌动蛋白的ESI-MS分析表明形成了一种蛋白质衍生物，其中有156Da(42028 Da)比天然肌动蛋白(41872 Da)增加了156Da，这与通过Michael加成加成一个HNE残基的情况一致。用NaBH4还原稳定G-肌动蛋白和F-肌动蛋白，并用胰酶消化，以确定HNE内收的位置。数据相关扫描模式下的LC-ESI-MS/MS分析结果明确地表明，Cys374是HNE加合的位置。计算研究表明，Cys374残基的反应性是由于Cys374周围特殊的微环境造成的显著的可及表面和大量的硫醇酸性。版权所有(C)2005 John Wiley&Sons，Ltd.
We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1: 1 to 1: 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH4 reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374. Copyright (c) 2005 John Wiley & Sons, Ltd.