DIRECT CHARACTERIZATION OF PROTEIN ADDUCTS OF THE LIPID-PEROXIDATION PRODUCT 4-HYDROXY-2-NONENAL USING ELECTROSPRAY MASS-SPECTROMETRY
DIRECT CHARACTERIZATION OF PROTEIN ADDUCTS OF THE LIPID-PEROXIDATION PRODUCT 4-HYDROXY-2-NONENAL USING ELECTROSPRAY MASS-SPECTROMETRY
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DOI：
10.1021/tx00046a009
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发表时间：
1995-06-01
影响因子：
4.1
通讯作者：
GERMAN, JB
中科院分区：
文献类型：
--
作者：
BRUENNER, BA;JONES, AD;GERMAN, JB
氧化应激和暴露于异生物质产生反应性物质，包括细胞毒性醛4-羟基-2-壬烯醛。这种醛表现出多种生物学效应，并已被报道为脂质过氧化的标志物。4-羟基-2-壬烯醛的毒性和致动脉粥样硬化性归因于共价蛋白加合物的形成。在当前的研究中，将两种模型蛋白质（β-乳球蛋白B和人血红蛋白）暴露于4-羟基-2-壬烯醛，并使用电喷雾电离质谱法对蛋白质加合物进行表征。我们的研究结果提供了明确和直接的证据，即>99%的蛋白质修饰是通过迈克尔加成发生的，并且仅形成痕量的席夫碱加合物。通过将修饰的蛋白质定量转化为肟和五氟苄基肟衍生物，获得了该结果的确认，如通过电喷雾电离质谱法、分光光度蛋白质羰基测定法和气相色谱/质谱法测定羟胺处理后释放的4-羟基-2-壬烯醛所证明的。这些结果进一步证明了蛋白质结合的醛对于后续反应或作为分子识别位点的可用性。迈克尔加成产物相对于席夫碱加合物的优势也表明，大多数测定生物组织或体液中4-羟基-2-壬烯醛的方法都是基于错误的假设，即肼或羟胺从蛋白质中释放4-羟基-2-壬烯醛。
Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that >99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.