Technology for evaluating drug-binding responses to small-molecule perturbation

项目摘要|摘要 蛋白质充当细胞的效应分子-执行大多数结构、调节和酶促作用。 功能协调发展的蛋白质本身通常通过与配体（包括金属）的直接相互作用来调节， 脂质、其他蛋白质和药物。这些蛋白质-配体相互作用是多种生物学的基础。 流程.然而，探索这些交互的技术在吞吐量和它们的性能方面都是有限的。 扩展能力。这些技术的局限性在一定程度上突出了近30年来， 蛋白质组学和基因组学技术的研究还不能完全表征 人类细胞中有两万个蛋白质编码基因。 为了解决这个问题，我们建议建立一个基石技术套件，用于高通量，蛋白质组范围内的 蛋白质-配体相互作用分析。在这项工作中，我们将展示一个 一种集中的方式来强调这种技术的潜力，为研究配体带来强大的定量方法 规模上的约束力。我们的技术创新集中在使用高通量方法检测蛋白质- 整个蛋白质组的配体相互作用。为此，我们将测量 通过与配体结合而诱导的蛋白质的热稳定性。我们测量这种热稳定性作为一个相对的 使用基于串联质量标签（TMT）的样品多路复用来测定蛋白质丰度的差异。样品 多路复用能够同时定量多达18个样品的蛋白质组。样品复用， TMT提高了样品吞吐量，减少了样品间的缺失值，并实现了复杂的 实验设计-例如，时间过程、剂量依赖性和敲除-挽救实验。 在拟议的工作过程中，我们将建立新的蛋白质组学技术， 蛋白质组范围的热稳定性测定和TMT定量以表征蛋白质-配体相互作用。的 （1）智能质谱数据采集，（2）蛋白质组热稳定性分析，和 (3)样品多路复用将使我们能够破译蛋白质和配体之间复杂的相互作用， 蛋白质组着眼于转化研究，我们将首先关注小分子药物， 配体，因为我们可以获得具有已知主要蛋白质靶标的不同文库。这些数据和方法将 用于揭示蛋白质组的配体扰动的功能和次级效应， 匹配整个蛋白质组和基因表达谱，以确定特定药物-蛋白质- 参与驱动细胞反应。

PROJECT SUMMARY | ABSTRACT Proteins act as the effector molecules of cells – carrying out most of the structural, regulatory, and enzymatic functions. Proteins themselves are often regulated through direct interaction with ligands, including metals, lipids, other proteins, and drugs. These protein-ligand interactions are fundamental to diverse biological processes. Yet, technologies to explore these interactions are limited in terms of both throughput and their ability to scale. The limits of these technologies are in part highlighted be the fact that for nearly 30 years, proteomics and genomics technologies research have been unable to fully characterize the functions of the 20,000 protein coding genes in human cells. To address this, we propose to build a cornerstone technology suite for high-throughput, proteome-wide protein-ligand interaction profiling. In this work we will demonstrate the development and implementation in a focused way to highlight the potential of this technology to bring robust quantitative approaches to study ligand binding at scale. Our technological innovations center on using high-throughput methods to detect protein- ligand interactions across the entire proteome in a single analysis. To do this, we will measure the change in thermal stability of proteins induced by binding to a ligand. We measure this thermal stability as a relative difference in protein abundance using sample multiplexing based on tandem mass tags (TMT). Sample multiplexing enables quantitation of up to 18 samples’ proteomes simultaneously. Sample multiplexing with TMT increases sample throughput, reduces missing values across samples, and enables complex experimental designs – e.g., time courses, dose dependency, and knockout-rescue experiments. Over the course of the proposed work, we will build new proteomics technologies to harness the benefits of proteome-wide thermal stability assays and TMT quantitation to characterize protein-ligand interactions. The combination of (1) intelligent mass spectrometric data acquisition, (2) proteome thermal stability profiling, and (3) sample multiplexing will enable us to decipher the complex interplay between proteins and ligands across the proteome. With an eye towards translational research, we will focus at first on small-molecule drugs as ligands as we can acquire diverse libraries with known primary protein targets. These data and methods will be used to reveal the functional and secondary effects of ligand perturbation of the proteome by leveraging matched whole proteome and gene expression profiles to determine to what extent specific drug-protein- engagement drives cellular responses.