School of Life and Health Sciences, Aston Triangle, Aston University, Birmingham, UK.
School of Life and Health Sciences, Aston Triangle, Aston University, Birmingham, UK.
Arch Biochem Biophys. 2018 May 15;646:38-45. doi: 10.1016/j.abb.2018.03.026. Epub 2018 Mar 23.
Lipids containing polyunsaturated fatty acids are primary targets of oxidation, which produces reactive short-chain aldehydes that can covalently modify proteins, a process called lipoxidation. Improved mass spectrometry (MS) methods for the analysis of these adducts in complex biological systems are needed. Lysozyme and human serum albumin (HSA) were used as model proteins to investigate lipoxidation products formed by two short-chain aldehydes, acrolein and pentanal, which are unsaturated and saturated aldehydes respectively. The adducts formed were stabilized by NaBH or NaBHCN reduction and analysed by MS. Analysis of intact modified lysozyme showed a pentanal modification resulting from Schiff's base formation (+70 Da), and up to 8 acrolein adducts, all resulting from Michael addition (+58 Da). Analysis of tryptic digests identified specific histidine, cysteine and lysine residues modified in both lysozyme and HSA, and determined characteristic amino acid-specific fragmentations. Eight different internal fragment ions were found that could be used as general diagnostic ions for pentanal- and acrolein-modified amino acids. The combined use of intact protein analysis and LC-MS/MS methods provided a powerful tool for the identification and localization of aldehyde-protein adducts, and the diagnostic ions will facilitate the development of targeted MS methods for analysis of adducts in more complex samples.
含有多不饱和脂肪酸的脂质是氧化的主要靶标，氧化会产生反应性的短链醛，这些醛可以与蛋白质发生共价修饰，这一过程称为脂质氧化。需要改进用于分析复杂生物体系中这些加合物的质谱 (MS) 方法。溶菌酶和人血清白蛋白 (HSA) 被用作模型蛋白，以研究两种短链醛（丙烯醛和戊醛）形成的脂质氧化产物，它们分别是不饱和醛和饱和醛。通过 NaBH 或 NaBHCN 还原稳定形成的加合物，并通过 MS 进行分析。对完整修饰的溶菌酶的分析表明，戊醛通过席夫碱形成（+70 Da）发生了修饰，并且多达 8 个丙烯醛加合物，全部通过迈克尔加成（+58 Da）形成。对胰蛋白酶消化产物的分析鉴定了在溶菌酶和 HSA 中均发生修饰的特定组氨酸、半胱氨酸和赖氨酸残基，并确定了特征性的氨基酸特异性片段。发现了 8 个不同的内部片段离子，可作为戊醛和丙烯醛修饰氨基酸的一般诊断离子。完整蛋白质分析与 LC-MS/MS 方法的结合为鉴定和定位醛-蛋白质加合物提供了有力工具，并且这些诊断离子将有助于开发用于分析更复杂样品中加合物的靶向 MS 方法。
