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

描述(申请人提供)：活性氧物种(ROS)与癌症的发生和发展，包括刺激肿瘤生长和转移，之间的关系已经确立；矛盾的是，ROS在许多涉及电离辐射和化疗的抗癌治疗中也是重要的参与者。然而，我们对ROS在许多正常和疾病相关的功能作用中所涉及的分子机制的认识有限，这主要是由于可用于研究ROS分子靶点的工具有限。我们在维克森林大学的研究团队在IMAT计划的支持下，率先开发了高度特异性的化学探针，可以检测和识别氧化蛋白质，目标是半胱氨酸硫醇氧化的初始亚磺酸(-SOH)产物。虽然这些探针已被成功地用于识别特定蛋白质内的氧化目标，例如Akt2(在上下文中 尽管它们与参与血管生成的特定脂筏相关蛋白酪氨酸磷酸酶(PDGF信号转导)和特定的脂筏相关蛋白酪氨酸磷酸酶有关，但它们尚未被证明能够用高通量质谱仪(MS)分析来大规模鉴定这些部位。正如我们的初步数据所显示的，干扰MS的因素已经被识别出来，并通过新的探针设计来规避；例如，这些干扰MS检测所需电荷状态的1，3-二羰基探针的酸碱性质可以通过产物的后标记环化来阻止，并且已经产生了与低丰度的磺酸具有更高反应活性的新的线性探针。本申请描述了额外的新策略，以克服限制氧化蛋白质组检测和分析的剩余问题。第一个目标是描述更有效地捕获亲电性和亲核性磺酸的新化学探针。对于第二个目标，我们将研究新的成像和MS技术，以原位显示选择性的蛋白质-SOH修饰，并识别内源性表达蛋白质中的磺酸位点。该项目的成功完成将产生很大的影响，使人们能够更深入地了解氧化还原控制的细胞内过程，涉及正常和癌症信号、血管生成和转移，以及化疗和基于辐射的治疗。从长远来看，它可能使设计选择性激动剂或拮抗剂来调节肿瘤中靶蛋白的活性成为可能。

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.