Development of New Proteomics Technology and its Application to Study Cellular Organization

蛋白质组学新技术的发展及其在细胞组织研究中的应用 我们实验室的广泛目标是获得对细胞组织的系统级别的了解，并开发 促进这项研究的蛋白质组学技术。多亏了人类基因组计划，我们有了几乎 组成细胞的所有分子的完整部件清单，但我们仍然很难理解这些分子是如何 走到一起，优雅地组织成一个有生命的系统。到目前为止，对这一组织的研究主要是通过 一次仔细观察一种蛋白质。虽然这种方法非常成功，但它不能 解决生物系统中更高水平的复杂性，这些复杂程度是由无数 组件。一次只看一个分子会严重阻碍对生物学的理解。相反，我们 一次调查整个系统。多重蛋白质组学的最新进展使我们能够观察到 在多种条件下，数千种蛋白质同时存在。结合经典生物化学 方法，我们可以揭示我们不会发现的集体行为和紧急性质 否则的话。我的实验室对系统级的蜂窝组织非常感兴趣。为了达到这个目标，这个提议 包含两个部分。 该提案的第一部分概述了我们打算如何改进定量蛋白质组学技术。 蛋白质组学已经变得非常强大。然而，在敏感性、数据质量、 和可访问性保持不变。我们努力解决这些问题。在过去的一年里，我们开发了一种新的 用于定量鸟枪式蛋白质组学(TMTproC)的方法，产生具有无与伦比的灵敏度和 测量质量的同时降低成本。接下来，我们的目标是使TMTproC与入门级质量兼容 光谱仪，这有可能使定量蛋白质组学民主化。此外，我们建议融合 具有数据独立采集(DIA)的TMTproC。我们预计，这将融合两者的好处 方法：一种用无限传递多重蛋白质组精致测量质量的方法 DIA的可扩展性。 第二部分描述了我们的目标是如何应用我们的技术进步来理解 系统层面的机制。首先，我们将通过以下方式集成被动扩散和主动交通模型 核孔来预测整个蛋白质组如何在细胞核和细胞质之间分割。第二，我们的目标 整合所有水平的蛋白质丰度，控制每个基因的转录、翻译和蛋白质 退化。我们将重点关注蛋白质周转率，这是这些参数中技术上最难测量的。 最终，我们的目标是确定作为受精卵的每个基因的蛋白质表达水平是如何被控制的。 发育成一个胚胎，有一个完全定义的身体计划。这些测量将为我们提供基本的 洞察发育中胚胎在健康和疾病中的调节和组织。

Development of New Proteomics Technology and its Application to Study Cellular Organization The broad goal of our lab is to obtain a systems-level understanding of cellular organization and develop proteomics technology that facilitates this research. Thanks to the human genome project, we have a nearly complete parts list of all molecules making up cells, but we still very poorly understand how these molecules come together and elegantly organize into a living system. So far, this organization has been studied mainly by looking carefully at one protein at a time. While this approach has been tremendously successful, it cannot address the higher levels of complexity in biological systems that arise from the interplay of a myriad of components. Looking at one molecule at a time can severely hinder understanding biology. Instead, we investigate the entire system all at once. Recent progress in multiplexed proteomics enables us to observe thousands of proteins simultaneously among multiple conditions. Combined with classical biochemical approaches, we can reveal collective behavior and emergent properties that we would not have discovered otherwise. My lab is broadly interested in systems-level cellular organization. Towards this goal, this proposal contains two parts. The first part of this proposal outlines how we intend to improve quantitative proteomics technology. Proteomics has become very powerful. Nevertheless, severe shortcomings concerning sensitivity, data quality, and accessibility remain. We strive to address these problems. Over the last year, we have developed a new method for quantitative shotgun proteomics (TMTproC), producing data with unmatched sensitivity and measurement quality while reducing cost. Next, we aim to make TMTproC compatible with entry-level mass spectrometers, which has the potential to democratize quantitative proteomics. Furthermore, we propose to fuse TMTproC with data-independent acquisition (DIA). We anticipate that this will fuse the benefits of both approaches: a method delivering the exquisite measurement quality of multiplexed proteomics with the infinite scalability of DIA. The second part describes how we aim to apply our technological advances toward understanding systems-level mechanisms. First, we will integrate passive diffusion and active transport models through the nuclear pore to predict how the entire proteome partitions between the nucleus and cytoplasm. Second, we aim to integrate all levels of protein abundance control aspects for every gene – transcription, translation, and protein degradation. We will focus on protein turnover, the technically most difficult to measure of these parameters. Ultimately, we aim to determine how protein expression levels are controlled for each gene as a fertilized zygote develops into an embryo with a fully defined body plan. These measurements will provide us with fundamental insight into the regulation and organization of developing embryos in health and disease.