Chemistry for next-generation single-molecule fluorosequencing technology 2.0.

项目摘要 人类蛋白质组极其复杂，包括> 10，000种蛋白质和100倍于每种蛋白质的蛋白质型。 基因产物在癌症和其他疾病中，几种新的蛋白质变体可能是由突变、融合 以及进一步影响蛋白质功能和结构的PTM。这就需要确定 蛋白质和PTM在细胞或生物体中的单分子水平，以了解生物过程， 疾病分析和生物标志物发现。尽管蛋白质测序在革命性的精确度方面的力量 在医学诊断中，没有单分子方法来鉴定蛋白质组中的蛋白质和PTM- 宽水平。因此，在理解蛋白质和PTM在生物学中的作用方面存在巨大差距， 疾病，由于缺乏有效的技术，分析低丰度蛋白质和PTM在一个单一的， 高度复杂的蛋白质组系统中的分子水平。这项研究的主要目的是填补 目前的差距，在一系列可用的技术测序和鉴定蛋白质和PTM在单一的- 分子水平。一套新的化学方法将被开发用于特定的侧链修饰， 氨基酸和PTM具有低反应性，因此难以修饰，以连接各种荧光 肽部分。作为一个训练有素的有机化学家和化学生物学家，并与 作为单分子蛋白质荧光测序的创始人（Eric Anslyn博士和艾德·马科特博士），我们处于领先地位 为了快速评估我们新开发的用于蛋白质组全分析的化学方法， 吞吐量的方式。高度的化学特异性和产率的新化学方法将避免 通过单分子荧光测序对氨基酸的下游错误鉴定。拟议研究 包含各种创新，用于推进单分子蛋白质测序。第一个创新，涉及 选择性标记甲基赖氨酸和甲基组氨酸PTM的化学方法，例如 （单甲基赖氨酸Kme、二甲基赖氨酸Kme 2、三甲基赖氨酸Kme 3和甲基组氨酸Hme）， 与单分子荧光测序兼容。第二个创新是化学的发展 用于选择性标记反应性较低的氨基酸如酰胺（Gln和Asn）、醚（Met）的方法， 和与单分子荧光测序相容的烷烃（Ile、Leu、瓦尔、Phe、Pro）。这些新 单分子荧光测序的化学方法将导致氨基酸和PTM的鉴定 具有高灵敏度、准确度和动态范围，能够在 以高通量的方式进行蛋白质组范围的规模。因此，拟议的研究具有很大的潜力， 进一步了解这些PTM如何调节各种细胞信号传导过程，并导致各种 疾病这些工具将导致发现新的甲基赖氨酸和甲基组氨酸生物标志物。这 这项研究还将使稀有蛋白质的检测成为可能，并可能揭示新的分子调控网络 从而为基础科学和医学诊断提供了前所未有的机会。

PROJECT SUMMARY The human proteome is extremely complex, comprising > 10,000 proteins and 100 times proteoforms for each gene product. In cancer and other diseases, several new protein variants may result from mutations, fusions and PTMs that further influence the functions and structure of proteins. This necessitates the identification of proteins and PTMs at a single-molecule level in a cell or an organism to understand biological processes, disease analysis and biomarker discovery. Despite the power of protein sequencing in revolutionizing precision medicine diagnostics, there are no single-molecule methods to identify proteins and PTMs at the proteome- wide level. Therefore, there is a huge gap in understanding the role of proteins and PTMs in biology and diseases due to the lack of efficient techniques for the analysis of low abundant proteins and PTMs at a single- molecule level in a highly complex proteome system. The main goal of this research proposal is to fill the present gap in the range of available techniques to sequence and identify proteins and PTMs at the single- molecule level. A new suite of chemical methods will be developed for specific modification of side chains of amino acids and PTMs that are of low reactivity thus challenging to modify, to attach various fluorescent moieties to peptides. As a trained organic chemist and chemical biologist, and in collaboration with the founders (Dr. Eric Anslyn and Dr. Ed Marcotte) of single-molecule protein fluorosequencing, we are positioned to rapidly evaluate our newly developed chemical methods for the proteome-wide analyses in a high throughput manner. A high degree of chemical specificity and yield of the new chemical methods will avoid downstream misidentification of amino acids by single-molecule fluorosequencing. The proposed research contains various innovations for advancing single-molecule protein sequencing. The First innovation, involves the chemical methods for the selective labeling of methyl lysine and methyl histidine PTMs, such as (monomethyl lysine Kme, dimethyl lysine Kme2, trimethyl lysine Kme3 and methylhistidine Hme) that are compatible with single molecule fluorosequencing. The second innovation is the development of chemical methods for the selective labeling of less reactive amino acids, such as amides (Gln and Asn), ethers (Met) and alkanes (Ile, Leu, Val, Phe, Pro) that are compatible with single molecule fluorosequencing. These new chemical methods for single molecule fluorosequencing will lead to the identification of amino acids and PTMs with high sensitivity, accuracy, and dynamic range capable of identifying low abundant proteins and PTMs at the proteome-wide scale in a high throughput manner. Thus, the proposed research has a great potential to further our understanding of how these PTMs regulate various cellular signaling processes and lead to various diseases. Such tools would lead to the discovery of novel methyl lysine and methyl histidine biomarkers. This research would also enable the detection of rare proteins and may uncover new molecular regulatory networks within cells thus opening unprecedented opportunities in basic science and medical diagnostics.