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

总结 泛素（Ub）通路的缺陷通常是癌症和破坏性神经退行性疾病的原因。 疾病由于它在生物回路中的核心作用和治疗干预的潜力， 系统是一个研究热点。然而，Ub生物学是高度复杂的，由超过500种Ub连接酶支持， 近100种去泛素化酶目前，这种复杂性和我们对Ub生物学玩家的有限了解， 它们之间的相互作用严重阻碍了对许多疾病进行有针对性的干预。考虑所有可能 为了解决这种复杂性，两种重要的方法具有从根本上解决问题的潜力。 彻底改变我们解开每种蛋白质在Ub生物学中独特的细胞作用的能力：i）样品多路复用， 基于质谱的蛋白质组学，以提高蛋白质组深度的通量，以及ii）化学 蛋白质组学，发现选择性的药理学工具来干扰和阐明功能。 通过上一个赠款周期内的努力，提高了样本复用水平， 16个样品的表达和修饰水平的蛋白质组范围的比较。此外，实时 数据库搜索使吞吐量加倍，同时提高了定量准确性。在这个提议中，我们现在把我们的 关注通过新的化学蛋白质组学发现研究Ub系统的化学探针 质谱分析已经是主流的工作流程。在目标1中，我们将创建两个下一代 工作流程-一个用于靶向，一个用于发现蛋白质组学-支持同量异位素标记研究， 16重试剂。重点将是使用外部控制器完全集成对扫描决策过程的控制。 软件和用于蛋白质定量的API，这将提高深度和定量精度。鉴于 大多数Ub系统酶中的反应中心是半胱氨酸（例如，E2，E3，去泛素化酶），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。研究在此 我们的目标是建立全面的蛋白质-小分子相互作用 在数千个分子的规模上，整个亲电化合物库的景观。我们的目标 本发明的目的是发现针对Ub连接酶和去泛素化酶的新探针。在目标3中，我们将创建工作流， 发现诱导Ub介导的蛋白质降解的小分子。我们将首先修改发现 和目标1的靶向平台，使我们能够从96孔培养的细胞中获得起始量， 筛选板。接下来，将开发基于平板的细胞测定来测量蛋白质组范围内的 在每个孔中用化合物池进行急性治疗的结果， 使Ub-蛋白酶体系统降解它们的靶标。实现这三个目标将提供： 创新的同量异位素标记研究的新工作流程，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，和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.