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

蛋白质组学新技术及其在细胞研究中的应用 组织 我们实验室的广泛目标是获得细胞组织的系统级理解， 发展蛋白质组学技术，促进这项研究。多亏了人类基因组 项目，我们有一个几乎完整的组成细胞的所有分子的零件清单，但我们仍然非常 我对这些分子是如何聚集在一起并有序地组织成一个生命体知之甚少 系统到目前为止，对这种组织的研究主要是通过仔细观察一种蛋白质， 时间虽然这一方法取得了巨大成功，但它不能解决更高的问题。 生物系统的复杂程度是由无数 件.一次只观察一个分子似乎严重阻碍了 了解生物学。相反，我们必须立即开始调查整个系统。 多重蛋白质组学的最新进展使我们能够观察成千上万的蛋白质 同时在多个条件下。结合经典的生物化学 这种方法使我们能够揭示集体行为和涌现特性， 没有机会发现其他的。我的实验室对系统级蜂窝网络 organization.为实现这一目标，本建议包括两个部分。第一部分描述了如何 我们的目标是破译蛋白质组如何在细胞核和细胞质之间分配， 发育和解码胚胎如何使用不同的核组成， 适当的转录时机和核大小控制。本建议的第二部分 概述了我们打算如何改进定量蛋白质组学技术。蛋白质组学已经成为 非常强大然而，在敏感性、数据质量和 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， 开发了一种新的定量鸟枪蛋白质组学（TMTc+）方法， 具有无与伦比的灵敏度和测量质量，同时降低了成本。接下来，我们的目标是 将联合收割机TMTc+与有针对性的方法相结合。我们预计，这将使我们能够量化 蛋白质下降到最低丰度的转录因子。这项新技术将使我们的 自己的研究细胞组织，但更普遍的将有利于研究人员， 对差异蛋白质表达水平感兴趣，以研究基础生物学或疾病。

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 near complete parts list of all molecules making up cells, but we still very poorly understand how all these molecules come together and organize marvelously into a living system. So far this organization has been mostly studied 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 arises from the interplay of a myriad of components. Looking at one molecule at a time seems to be serious hindrance towards understanding biology. Rather, we have to start investigating the entire system all at once. Recent progress in multiplexed proteomics enables us to observe thousands of proteins simultaneously among multiple conditions. In combination with classical biochemical approaches this allows us to reveal collective behavior and emergent properties we would have no chance to discover otherwise. My lab is broadly interested in systems-level cellular organization. Towards this goal, this proposal contains two parts. The first part describes how we aim to decipher how the proteome partitions between nucleus and cytoplasm in early development and decode how the embryos uses differential nuclear composition for the appropriate timing of transcription and nuclear size control. The second part of this proposal outlines how we intend to improve quantitative proteomics technology. Proteomics has become very powerful. Nevertheless, severe shortcomings in respect to 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 (TMTc+), which produces data with unmatched sensitivity and measurement quality while reducing cost. Next, we aim to combine TMTc+ with a targeted approach. We anticipate that this will enable us to quantify proteins down to the lowest abundant transcription factors. This new technology will benefit our own research of cellular organization, but more generally will benefit researchers that are interested in differential protein expression levels to study basic biology or disease.