Project 4: Protein adduction by electrophilic products of lipid oxidation

在PPG的初始资助期间，我们和我们的同事实施了质谱分析， （MS）为基础的蛋白质组学技术和新的亲和标记和捕获技术，使 选择性捕获和分析由特定脂质氧化产物标记的蛋白质。我们的整体目标 在这一竞争性的更新申请中， 和血浆，并应用灵敏的MS和免疫亲和方法来研究蛋白加合物作为调节剂 氧化应激的生物学反应和氧化应激的生物标志物。一个主要的推力 建议的工作是评估脂质亲电体在这两种情况下的目标选择性和生物效应。 它们的“游离”和磷脂酯化形式。这些研究将共同促进我们对 脂质亲电体对蛋白质损伤如何将环境诱导的氧化应激转化为离散的 信号传导效应，并将识别血浆和组织中氧化损伤的新候选生物标志物。 项目4的具体目标是：1）在细胞模型中鉴定脂质亲电体的蛋白质靶点。 这些研究将使用基于点击化学的方法来表征脂质亲电体的蛋白加合 来捕获和分析蛋白质和肽加合物。2)为了识别潜在的损伤敏感细胞， 通过将亲电加合的蛋白质映射到细胞 过程，经典途径，蛋白质-蛋白质相互作用网络存档在本地数据库中。3)到 鉴定调节氧化应激反应的蛋白质靶点和蛋白质上相应的加合位点 细胞内的压力。这项工作将与项目3合作进行，以测试以下假设： 脂质亲电体的特异性蛋白质触发信号传导和转录因子调节的变化 与压力有关。4)评价载脂蛋白A1（ApoA 1）加合物作为氧化应激的生物标志物 活的我们将评估ApoA 1亲核试剂与脂质亲电试剂的分级反应性， 亲电探针和内源性HDL脂质体外血浆氧化。多反应监测 将针对特定加合物开发基于MRM的LC-MS-MS定量分析，并将对其进行评价 与项目2合作，在人体样本中作为生物标志物候选物。

During the initial funding period of this PPG, we and our colleagues have implemented mass spectrometry (MS)-based proteomics technologies and novel affinity labeling and capture techniques to enable the selective capture and analysis of proteins labeled by specific lipid oxidation products. Our overall objective in this competing renewal application is to define the scope of protein damage by lipid electrophiles in cells and plasma and to apply sensitive MS and immunoaffinity methods to study protein adducts as modulators of biological responses to oxidative stress and as biomarkers for oxidative stress. A major thrust of the proposed work is an evaluation of the target selectivity and biological effects of lipid electrophiles in both their "free" and phospholipid-esterified forms. These studies will collectively advance our understanding of how protein damage by lipid electrophiles transduces environmentally-induced oxidatve stress into discrete signaling effects and will identify new candidate biomarkers for oxidative damage in plasma and tissues. The specific aims of Project 4 are: 1) To identify the protein targets of lipid electrophiles in cell models. These studies will characterize protein adduction by lipid electrophiles using Click chemistry-based methods to capture and analyze protein and peptide adducts. 2) To identify potential damage-susceptible cellular systems and candidate electrophile sensors by mapping electrophile-adducted proteins to cellular processes, canonical pathways, and protein-protein interaction networks archived in a local database. 3) To identify protein targets and corresponding adduction sites on proteins that regulate responses to oxidative stress in cells. This work will be done in collaboration with Project 3 to test the hypothesis that alkylation of specific proteins by lipid electrophiles triggers signaling and transcription factor regulation changes associated with stress. 4) To evaluate apolipoprotein A1 (ApoA1) adducts as biomarkers of oxidative stress 'n vivo. We will evaluate the hierarchical reactivity of ApoA1 nucleophiles with lipid electrophiles from electrophile probes and endogenous HDL lipids in plasma oxidations in vitro. Multiple reaction monitoring (MRM)-based LC-MS-MS quantitative assays will be developed for specific adducts, which will be evaluated as biomarker candidates in vivo in human specimens in collaboration with Project 2.