Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal
Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal
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DOI：
10.1002/jms.1067
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发表时间：
2006-09-01
影响因子：
2.3
通讯作者：
Carini, Marina
中科院分区：
文献类型：
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作者：
Aldini, Giancarlo;Gamberoni, Luca;Carini, Marina
几项证据表明，HNE修饰的白蛋白是一种有前途的全身性氧化应激的生物标志物，HNE修饰的白蛋白可能有助于脂质过氧化衍生抗原引发的免疫反应。在这项研究中，我们通过HPLC分析发现，HNE被人血清白蛋白（HSA）迅速淬灭，因为共价加合到蛋白质的不同可接近的亲核残基，如通过电喷雾电离质谱（ESI-MS）直接输注实验（1至9个HNE加合物，取决于所用的摩尔比，从1：0.25到1：5 HSA：HNE）所证明的。然后将LC-ESI-MS/MS方法应用于酶消化的HNE修饰的白蛋白，其允许鉴定11种不同的HNE加合物，8种迈克尔加合物（MA）和3种希夫碱（SB），涉及9个亲核位点，即：His67（MA）、His146（MA）、His242（MA）、His288（MA）、His510（MA）、Lys195（SB）、Lys199（MA，SB）、Lys525（MA，SB）和Cys34（MA）。发现最具反应性的HNE-加合位点是Cys 34（MA），其次是Lys 199，其主要通过形成席夫碱和His 146反应，得到相应的HNE迈克尔加合物。这些白蛋白修饰是HNE加合白蛋白的合适标签，并且可能是人体中氧化和羰基化损伤的有用生物标志物。版权所有（c）2006约翰威利父子有限公司。
Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1: 0.25 to 1: 5 HSA:HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright (c) 2006 John Wiley & Sons, Ltd.