New Reagents for Tracking Protein Oxidation in Cells by MS and Imaging Methods

通过质谱和成像方法追踪细胞中蛋白质氧化的新试剂

基本信息

批准号：
8547235
负责人：
Cristina Maria Furdui
金额：
$ 34.21万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8547235
关键词：
Acids Agonist Amino Acids Antineoplastic Agents Biochemistry Biological Assay Biological Markers Biotin Blood Vessels Case Study Cell Survival Cells Charge Chemicals Chemistry Chronic Communities Cyclization Cysteine DNA Data Detection Development Diet Disease Disease Progression Drug Design Environmental Pollutants Event Exhibits Fluorescent Dyes Generations Glutathione Goals Growth Human Image Imagery In Situ Infection Inflammation Intervention Investigation Ionizing radiation Label Lead Ligation Location Malignant - descriptor Malignant Neoplasms Mass Spectrum Analysis Matrix Metalloproteinases Mediating Membrane Microdomains Metabolic Methods Modification Molecular Molecular Medicine Molecular Target Monitor Neoplasm Metastasis Oligonucleotides Organelles Oxidation-Reduction Pathway interactions Peptides Pharmaceutical Preparations Phosphorylation Phosphorylation Site Platelet-Derived Growth Factor Play Post-Translational Protein Processing Process Property Protein Isoforms Protein Tyrosine Phosphatase Proteins Proteome Publications Radiation Reactive Oxygen Species Reagent Recombinants Reducing Agents Reporter Reporting Research Research Design Research Personnel Role Sampling Signal Transduction Site Study models Sulfenic Acids Sulfhydryl Compounds TEV protease Technology Testing Time Tissue Sample Toxic Environmental Substances Universities Virus angiogenesis base cancer cell cancer therapy cellular imaging chemotherapy design dicyanmethane disulfide bond forest genetic regulatory protein imaging modality improved killings method development oxidation pathogenic bacteria programs protein function public health relevance response single molecule tool trafficking tumor tumor growth tumor progression

项目摘要

DESCRIPTION (provided by applicant): The association of reactive oxygen species (ROS) with the initiation and progression of cancer, including stimulation of tumor growth and metastasis, is well established; paradoxically, ROS are also important players in many anti-cancer treatments involving ionizing radiation and chemotherapies. Yet we have only a limited appreciation for the molecular mechanisms involved in the many normal and disease-associated functional roles played by ROS, largely due to the limited tools available for studying the molecular targets of ROS. Our research team at Wake Forest University has pioneered the development of highly specific chemical probes, with previous support from the IMAT program, which enable detection and identification of oxidized proteins, targeting the initial sulfenic acid(-SOH) product of cysteine thiols undergoing oxidation. While these probes have been used successfully to identify targets of oxidation within specific proteins such as Akt2 (in the context of PDGF signaling) and specific lipid raft-associated protein tyrosine phosphatases involved in angiogenesis, they have not yet proven amenable to wide-scale identification of such sites using high- throughput mass spectrometry (MS) analysis. As demonstrated in our preliminary data, factors which interfere with MS have been identified and circumvented with new probe designs; for example, acid-base properties of these 1,3-dicarbonyl probes which interfere with the charge states needed for MS detection can be blocked by post-labeling cyclization of the products, and new linear probes exhibiting much higher reactivity with the low abundance sulfenic acids have been generated. This application describes additional new strategies to overcome the remaining issues that limit detection and analysis of the oxidized proteome. The first aim describes new chemical probes for more efficient trapping of electrophilic and nucleophilic sulfenic acids. With the second aim we will investigate new imaging and MS technologies to visualize selective protein -SOH modification in situ and identify sulfenic acid sites in endogenously expressed proteins. Successful completion of this project will have high impact, enabling a much deeper understanding of redox-controlled intracellular processes involved in normal and cancer signaling, angiogenesis and metastasis, as well as chemotherapeutic and radiation-based treatments. In the long term, it may enable the design of selective agonists or antagonists to modulate the activity of target proteins in tumors.

描述（由申请人提供）：活性氧（ROS）与癌症的发生和进展（包括刺激肿瘤生长和转移）之间的关系已得到充分证实；矛盾的是，ROS 在许多涉及电离辐射和化疗的抗癌治疗中也发挥着重要作用。然而，我们对 ROS 所发挥的许多正常和疾病相关功能作用的分子机制了解有限，这很大程度上是由于可用于研究 ROS 分子靶点的工具有限。我们维克森林大学的研究团队在 IMAT 项目的先前支持下，率先开发了高度特异性的化学探针，该探针能够检测和识别氧化蛋白质，针对氧化过程中半胱氨酸硫醇的初始亚磺酸 (-SOH) 产物。虽然这些探针已成功用于识别特定蛋白质（例如 Akt2）内的氧化靶标（在上下文中） PDGF信号传导）和参与血管生成的特定脂筏相关蛋白酪氨酸磷酸酶，尚未证明它们适合使用高通量质谱（MS）分析来大规模鉴定此类位点。正如我们的初步数据所示，干扰 MS 的因素已被识别并通过新的探针设计规避；例如，这些 1,3-二羰基探针的酸碱特性会干扰 MS 检测所需的电荷态，可以通过产物的标记后环化来阻断，并且新的线性探针与低丰度的硫基表现出更高的反应性已产生酸。该申请描述了额外的新策略来克服限制氧化蛋白质组检测和分析的剩余问题。第一个目标描述了用于更有效地捕获亲电和亲核次磺酸的新化学探针。第二个目标是，我们将研究新的成像和 MS 技术，以可视化原位选择性蛋白质 -SOH 修饰并识别内源表达蛋白质中的次磺酸位点。该项目的成功完成将产生重大影响，使人们能够更深入地了解参与正常和癌症信号传导、血管生成和转移以及化疗和放射治疗的氧化还原控制的细胞内过程。从长远来看，它可能能够设计选择性激动剂或拮抗剂来调节肿瘤中靶蛋白的活性。