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

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）的相关性已得到充分证实;矛盾的是，ROS在许多涉及电离辐射和化疗的抗癌治疗中也是重要的参与者。然而，我们只有一个有限的欣赏的分子机制，参与许多正常和疾病相关的功能作用的ROS，主要是由于有限的工具，可用于研究ROS的分子靶点。我们在维克森林大学的研究团队率先开发了高度特异性的化学探针，之前得到了IMAT计划的支持，该计划能够检测和鉴定氧化蛋白质，目标是半胱氨酸硫醇氧化的初始次磺酸（-SOH）产物。虽然这些探针已成功地用于鉴定特定蛋白质如Akt 2内的氧化靶标（在本文中， 的PDGF信号传导）和参与血管生成的特异性脂筏相关蛋白酪氨酸磷酸酶，但它们尚未被证明适用于使用高通量质谱（MS）分析来大规模鉴定这些位点。如我们的初步数据所示，干扰MS的因素已被确定并通过新的探针设计规避;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通过产物的后标记环化来阻断，并且已产生了与低丰度次磺酸表现出高得多的反应性的新线性探针。本申请描述了另外的新策略，以克服限制氧化蛋白质组的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，我们将研究新的成像和MS技术，以可视化选择性的蛋白质-SOH修饰原位和确定在内源性表达的蛋白质中的次磺酸位点。该项目的成功完成将产生很大的影响，使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有更深入的了解。从长远来看，它可能使选择性激动剂或拮抗剂的设计，以调节肿瘤中的靶蛋白的活性。

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.