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首页> 外文学位 >Mass spectrometry-based identification and characterization of protein and peptide adducts of lipoxidation-derived aldehydes.
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Mass spectrometry-based identification and characterization of protein and peptide adducts of lipoxidation-derived aldehydes.

机译：基于质谱的脂氧化衍生醛蛋白和肽加合物的鉴定和表征。

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摘要

Oxidative stress is recognized as an important underlying factor in the pathogenesis of many degenerative diseases as well as normal senescence. The free radicals, reactive oxygen species (ROS) and electrophiles produced during oxidative stress are capable of modifying nucleic acids, lipids and proteins. There are a variety of oxidative modifications that occur to proteins including: cleavage of the protein backbone, direct oxidation of amino acid side chains by ROS, and adduction by electrophilic species such as lipid peroxidation products. Many of these oxidative modifications result in the introduction of carbonyl groups into the proteins. Protein carbonylation levels are commonly used as a biomarker to assess the degree of oxidative damage to a system. However the most commonly employed methods for measuring oxidative modifications to proteins, typically fail to provide any information about the identity of the modified protein, site of modification, or the chemical nature of the modification.;In the present study we develop an analytical technique based on affinity labeling with N'-aminooxymethylcarbonylhydrazino-D-biotin (aldehyde reactive probe, ARP), along with mass spectrometric analysis which allows for the full characterization of protein carbonylation modifications. The ability of the ARP method was first demonstrated for the case of oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions with alpha,beta-unsaturated aldehydic lipid peroxidation products with nucleophilic amino acid residue side chains. ARP was used to label a 4-hydroxy-2-nonenal (HNE) modified cysteine containing model peptide, and HNE modified E. coli thioredoxin, which were characterized using ESI-MS/MS and MALDI-MS/MS. ARP was also used to label the oxidative modifications alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS), formed during the metal catalyzed oxidation of GAPDH.;After demonstrating the utility of the technique on model systems, it was then applied to complex biological systems. In one case, subsarcolemmal mitochondria (SSM) isolated from rat cardiac tissue. Mitochondria are well known to be a major source of ROS within the cell. They are therefore important mediators of oxidative stress, as well as regulators of cell death. We were able to identify 39 unique sites on 27 mitochondrial proteins which were modified by six different alpha,beta-unsaturated aldehydes, including acrolein, beta-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Additionally we identified nine Lys residues on four mitochondrial proteins that were oxidized to AAS and subsequently labeled with ARP. The proteins identified with oxidative modifications include members of the mitochondrial electron transport chain, TCA cycle, membrane transport, lipid metabolism, and other important mitochondrial enzymes.;The ARP technique was also applied to identify protein targets of 4-hyroxy-2-nonenal in human monocytic THP-1 cells that were exogenously exposed to HNE. It was shown previously that exposure of THP-1 cells to HNE resulted in apoptosis, necrosis and protein carbonylation. We applied a multi-pronged proteomic approach involving electrophoretic, immunoblotting and mass spectrometric analysis to unequivocally identify eighteen sites of HNE modification on sixteen proteins. It was also demonstrated in this study that pretreatment of THP-1 cells with ascorbic acid resulted in decreased levels of HNE-protein conjugate formation.
机译：氧化应激被认为是许多退行性疾病以及正常衰老的发病机理中的重要基础因素。在氧化应激过程中产生的自由基，活性氧（ROS）和亲电试剂能够修饰核酸，脂质和蛋白质。蛋白质会发生多种氧化修饰，包括：蛋白质骨架的切割，ROS对氨基酸侧链的直接氧化以及诸如脂质过氧化产物之类的亲电物质的加成。这些氧化修饰中的许多导致将羰基引入蛋白质中。蛋白质羰基化水平通常用作评估系统氧化损伤程度的生物标记。然而，最常用的测量蛋白质氧化修饰的方法通常无法提供有关修饰蛋白质的身份，修饰位点或修饰化学性质的任何信息。在本研究中，我们开发了一种基于N'-氨基氧基甲基羰基肼基-D-生物素（醛反应性探针，ARP）进行亲和标记的方法，以及质谱分析，可对蛋白质羰基化修饰进行全面表征。 ARP方法的能力首次证明是通过迈克尔加成型偶联反应与具有亲核氨基酸残基侧链的α，β-不饱和醛脂质过氧化产物形成的脂质肽和蛋白质偶联物而实现的。 ARP用来标记含4-羟基-2-壬烯醛（HNE）修饰的半胱氨酸模型肽和HNE修饰的大肠杆菌硫氧还蛋白，使用ESI-MS / MS和MALDI-MS / MS对其进行表征。 ARP还用于标记在金属催化的GAPDH氧化过程中形成的氧化修饰的α-氨基己二酸半醛（AAS）和γ-谷氨酸半醛（GGS）;在证明该技术在模型系统上的用途后，将其应用到复杂的生物系统。在一种情况下，从大鼠心脏组织中分离出了肌膜下线粒体（SSM）。众所周知，线粒体是细胞内ROS的主要来源。因此，它们是氧化应激的重要介体，也是细胞死亡的调节剂。我们能够在27个线粒体蛋白上鉴定39个独特位点，这些位点被6种不同的α，β-不饱和醛修饰，包括丙烯醛，β-羟基丙烯醛，巴豆醛，4-羟基-2-己烯醛，4-羟基-2-壬烯醛和4-氧-2-壬烯此外，我们在四个线粒体蛋白上鉴定出9个Lys残基，这些残基被氧化为AAS，随后用ARP标记。被氧化修饰的蛋白质包括线粒体电子转运链，TCA循环，膜转运，脂质代谢和其他重要的线粒体酶。ARP技术还被用于鉴定4-羟基-2-壬烯醛的蛋白质靶标。外源性暴露于HNE的人单核THP-1细胞。先前已证明，THP-1细胞暴露于HNE会导致凋亡，坏死和蛋白质羰基化。我们应用了包括电泳，免疫印迹和质谱分析在内的多管脚蛋白质组学方法，明确鉴定了16种蛋白质上HNE修饰的18个位点。在这项研究中还证明了用抗坏血酸预处理THP-1细胞会导致HNE-蛋白质缀合物形成水平降低。

著录项

  • 作者

    Chavez, Juan D.;

  • 作者单位

    Oregon State University.;

  • 授予单位 Oregon State University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 375 p.
  • 总页数 375
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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