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

• 批准号: 10623824
• 负责人: Martin Wühr
• 金额: $ 43.74万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623824
• 关键词: Active Biological Transport; Address; Behavior; Biochemical; Biology; Cell Nucleus; Cells; Cytoplasm; Data; Democracy; Development; Diffusion; Disease; Embryo; Genes; Genetic Transcription; Goals; Health; Human Genome Project; Measurement; Measures; Methods; Modeling; Nuclear Pore; Organism; Property; Proteins; Proteome; Proteomics; Regulation; Research; Shotguns; System; Techniques; Technology; Time; Translations; biological systems; cost; data quality; experimental study; improved; insight; interest; mass spectrometer; new technology; protein degradation; protein expression; zygote

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.

蛋白质组学新技术的发展及其在细胞组织研究中的应用 我们实验室的主要目标是获得细胞组织的系统级理解，并开发 蛋白质组学技术促进了这项研究。多亏了人类基因组计划，我们有了一个几乎 完整的组成细胞的所有分子的部件列表，但我们仍然非常不了解这些分子如何 聚集在一起，优雅地组织成一个有生命的系统。到目前为止，这个组织主要是由 一次仔细观察一种蛋白质。虽然这种方法取得了巨大的成功，但它不能 解决生物系统中更高层次的复杂性，这些复杂性来自于无数 件.一次只看一个分子会严重阻碍对生物学的理解。而是 同时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 在多种条件下同时产生数千种蛋白质。结合经典的生物化学 方法，我们可以揭示集体行为和紧急性质，我们不会发现 否则我的实验室对系统级的细胞组织感兴趣。为了实现这一目标，该提案 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、 可访问性仍然存在。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 定量鸟枪蛋白质组学（TMTproC）方法，产生具有无与伦比灵敏度的数据， 测量质量，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，我们建议将 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：一种提供多重蛋白质组学的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将我们的技术进步应用于理解 系统级机制。首先，我们将通过 核孔来预测整个蛋白质组如何在细胞核和细胞质之间分配。第二，我们的目标 整合每个基因的所有水平的蛋白质丰度控制方面-转录，翻译和蛋白质 降解我们将关注蛋白质周转，这是这些参数中技术上最难测量的。 最终，我们的目标是确定蛋白质表达水平是如何控制每个基因作为一个受精卵 发育成一个有着完整体型的胚胎。这些测量将为我们提供基本的 深入了解健康和疾病中发育胚胎的调节和组织。

项目成果

Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery.

• DOI: 10.1016/j.cell.2023.11.007
• 发表时间: 2023-12-07
• 期刊: CELL
• 影响因子: 64.5
• 作者: Kafri, Moshe;Patena, Weronika;Martin, Lance;Wang, Lianyong;Gomer, Gillian;Ergun, Sabrina L.;Sirkejyan, Arthur K.;Goh, Audrey;Wilson, Alexandra T.;Gavrilenko, Sophia E.;Breker, Michal;Roichman, Asael;McWhite, Claire D.;Rabinowitz, Joshua D.;Cross, Frederick R.;Wuhr, Martin;Jonikas, Martin C.
• 通讯作者: Jonikas, Martin C.

Evaluating the Arrhenius equation for developmental processes.

• DOI: 10.15252/msb.20209895
• 发表时间: 2021-08
• 期刊: Molecular systems biology
• 影响因子: 9.9
• 作者: Crapse J;Pappireddi N;Gupta M;Shvartsman SY;Wieschaus E;Wühr M
• 通讯作者: Wühr M

Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.

超分辨率质谱可在 MS2 水平上实现快速、准确和高度多重的蛋白质组学。

• DOI: 10.1021/acs.analchem.2c04742
• 发表时间: 2023
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Kozhinov,AntonN;Johnson,Alex;Nagornov,KonstantinO;Stadlmeier,Michael;Martin,WarhamLance;Dayon,Loïc;Corthésy,John;Wühr,Martin;Tsybin,YuryO
• 通讯作者: Tsybin,YuryO

GCN2 adapts protein synthesis to scavenging-dependent growth.

• DOI: 10.1016/j.cels.2021.09.014
• 发表时间: 2022-02-16
• 期刊: Cell systems
• 影响因子: 9.3
• 作者: Nofal M;Wang T;Yang L;Jankowski CSR;Hsin-Jung Li S;Han S;Parsons L;Frese AN;Gitai Z;Anthony TG;Wühr M;Sabatini DM;Rabinowitz JD
• 通讯作者: Rabinowitz JD

Evidence for widespread cytoplasmic structuring into mesoscale condensates.

• DOI: 10.1038/s41556-024-01363-5
• 发表时间: 2024-02
• 期刊: Nature cell biology
• 影响因子: 21.3
• 作者: Felix C Keber;Thao Nguyen;Andrea Mariossi;C. Brangwynne;M. Wühr