New Sample Multiplexing Technologies to Identify Chemical Probes and Illuminate Ubiquitin Biology

摘要 泛素(Ub)途径的缺陷常常导致癌症和毁灭性的神经退行性变 疾病。由于它在生物回路中的核心作用和治疗干预的潜力，Ub 系统是一个非常热门的研究领域。然而，Ub生物学是高度复杂的，由500多个Ub连接酶和 将近100种去泛素酶。目前，这种复杂性和我们对Ub生物玩家和 它们的相互作用严重阻碍了许多疾病的靶向干预。考虑到所有可能 解决这种复杂性的方法，有两种重要的方法有很强的潜力从根本上 彻底改变我们解开每种蛋白质在Ub生物学中独特细胞角色的能力：i)样本多路传输 基于质谱学的蛋白质组学，以提高蛋白质组宽深度的吞吐量，以及ii)化学 蛋白质组学，以发现干扰和阐明功能的选择性药理工具。 通过在前一个赠款周期内的努力，提高了样本多路复用的水平，使 对16个样本的表达和修饰水平进行蛋白质组学比较。此外，一个实时的 数据库搜索使吞吐量翻了一番，同时提高了定量准确性。在这项提案中，我们现在将我们的 关注通过新的化学蛋白质组学发现化学探针来研究Ub系统 质谱学已经成为主流的工作流程。在目标1中，我们将创建两个下一代 工作流-一个用于靶向蛋白质组学，一个用于发现蛋白质组学-支持等压标记研究 16种复合试剂。重点将放在使用外部设备完全集成对扫描决策过程的控制 用于蛋白质定量的软件和API，这将提高深度和定量精度。考虑到 大多数Ub系统酶中的反应中心是半胱氨酸(例如，E2s、E3s、去泛素酶)，在目标2中，我们将 修改AIM 1工作流程，以允许在化学蛋白质组学中进行反应性半胱氨酸分析。在此基础上的研究 目标的完成将着眼于建立全面的蛋白质-小分子相互作用 数以千计的分子规模的亲电化合物的整个图书馆的景观。我们的目标是 本研究的目的是发现针对Ub连接酶和脱泛素酶的新型探针。在Aim 3中，我们将创建工作流以 发现诱导Ub介导的蛋白质降解的小分子。我们将首先修改这两个发现 和来自Aim 1的目标平台，使我们能够使用在96孔中生长的细胞的起始量 放映板。下一步，将开发一种基于平板的细胞分析来测量整个蛋白质组 在每口井中使用化合物池进行急性治疗的后果，准确地指出直接 使用Ub-蛋白酶体系统来降低他们的目标。为实现这三个目标，我将提供 创新的等压标记研究新工作流程，ii)产生新的化学探针作为研究 Ub在细胞过程中的不同作用，以及iii)为治疗干预创造新的前体。

Summary Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub system is an intense research area. Yet, Ub biology is highly complex, supported by over 500 Ub ligases and nearly 100 deubiquitinases. Currently, this complexity and our limited understanding of Ub biology players and their interactions are severe hindrances for targeted intervention in many diseases. Considering all possible ways to address this complexity, two important approaches have strong potential to fundamentally revolutionize our ability to unravel each protein's unique cellular role in Ub biology: i) sample multiplexing in mass spectrometry-based proteomics to improve throughput at proteome-wide depth, and ii) chemical proteomics to discover selective pharmacological tools to perturb and illuminate function. Through efforts within the previous grant cycle, the level of sample multiplexing was increased, allowing the proteome-wide comparison of expression and modification levels for 16 samples. In addition, a real-time database search doubled throughput while improving quantitative accuracy. In this proposal, we now turn our attention towards discovering chemical probes to study the Ub system through new chemical proteomics workflows where mass spectrometry is already a mainstay. In Aim 1, we will create two next-generation workflows—one for targeted and one for discovery proteomics—supporting isobaric tagging studies with 16plex reagents. The focus will be on fully integrating control over the scan decision process using external software and an API for protein quantification which will improve depth and quantitative precision. Given that reaction centers in most Ub system enzymes are cysteines (e.g., E2s, E3s, deubiquitinases), in Aim 2, we will modify the Aim 1 workflows to allow reactive cysteine profiling in chemical proteomics. Research under this aim will be completed with an eye toward establishing comprehensive protein-small molecule interaction landscapes for entire libraries of electrophilic compounds on a scale of thousands of molecules. Our goal in this Aim is to discover novel probes to Ub ligases and deubiquitinases. In Aim 3, we will create workflows to discover small molecules that induce Ub-mediated protein degradation. We will first modify both the discovery and targeted platforms from Aim 1 to allow us to work with starting amounts from cells growing in 96-well screening plates. Next, a plate-based cellular assay will be developed to measure the proteome-wide consequences of acute treatment with pools of compounds in each well, pinpointing compounds that directly engage the Ub-proteasome system to degrade their targets. The realization of these three aims will i) provide innovative new workflows for isobaric tagging studies, ii) generate new chemical probes as tools to study the diverse roles of Ub in cellular processes, and iii) create new precursors for therapeutic intervention.