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：
8721898
负责人：
Cristina Maria Furdui
金额：
$ 31.1万
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/8721898
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）：充分性氧（ROS）与癌症的起始和进展（包括肿瘤生长和转移的刺激）的结合；矛盾的是，ROS在许多涉及电离辐射和化学疗法的抗癌治疗中也是重要的参与者。然而，我们对ROS扮演的许多正常和疾病相关的功能作用的分子机制的欣赏仅有限，这在很大程度上是由于可用于研究ROS的分子靶标的有限的工具。在IMAT计划的先前支持下，Wake Forest University的研究团队率先开发了高度特异性的化学探针，该探测器可以检测和鉴定氧化蛋白质，以氧化氧化的初始硫酸（-SOH）乘积。尽管这些探针已成功地用于识别特定蛋白质（例如Akt2）内的氧化靶 PDGF信号传导）和参与血管生成的特定脂质筏相关蛋白酪氨酸磷酸酶，它们尚未证明使用高吞吐量质谱法（MS）分析可与此类部位的广泛鉴定。正如我们的初步数据所示，干扰MS的因素已被鉴定出来并通过新的探针设计进行了规避。例如，这些1,3-二甲苯探针的酸碱特性可以通过产物后标记的自环化来阻止MS检测所需的电荷状态，而新的线性探针具有更高的反应性，其反应性高得多酸已经产生。该应用程序描述了克服限制氧化蛋白质组检测和分析的其余问题的其他新策略。第一个目的描述了新的化学探针，以更有效地捕获亲电和亲核硫酸。以第二个目的，我们将研究新的成像和MS技术，以可视化选择性蛋白质-SOH原位修饰，并在内源表达的蛋白质中鉴定硫酸位点。该项目的成功完成将产生很大的影响，从而使人们对参与正常和癌症信号，血管生成和转移的氧化还原控制的细胞内过程以及化学治疗和基于辐射的治疗。从长远来看，它可以使选择性激动剂或拮抗剂的设计能够调节肿瘤中靶蛋白的活性。