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

项目总结 人类蛋白质组极其复杂，由10,000种蛋白质组成，每种蛋白质都有100倍的蛋白质形式 基因产物。在癌症和其他疾病中，几种新的蛋白质变异可能是由于突变、融合 和PTM，进一步影响蛋白质的功能和结构。这就需要确定 在细胞或生物体中的单分子水平上的蛋白质和PTM以了解生物过程， 疾病分析和生物标志物的发现。尽管蛋白质测序具有革命性的精确度 医学诊断学，目前还没有单分子方法来识别蛋白质组中的蛋白质和PTM- 宽阔的水平。因此，在理解蛋白质和PTM在生物学和生物学中的作用方面存在着巨大的差距。 由于缺乏有效的技术来分析低丰度的蛋白质和PTM而导致的疾病. 在高度复杂的蛋白质组系统中的分子水平。这项研究提案的主要目标是填补 目前在对蛋白质和PTM进行测序和鉴定的现有技术范围内存在差距- 分子水平。将开发一套新的化学方法来对侧链进行特定修饰 低反应性的氨基酸和PTM，因此很难修饰、结合各种荧光 多肽的一部分。作为训练有素的有机化学家和化学生物学家，并与 单分子蛋白质荧光测序的创始人(埃里克·安斯林博士和埃德·马科特博士)，我们处于 快速评估我们最新开发的蛋白质组全分析化学方法 吞吐量方式。新的化学方法的高度化学专一性和产率将避免 单分子荧光测序法对氨基酸下游错误识别的研究。拟议的研究 包含推进单分子蛋白质测序的各种创新。第一项创新，涉及 选择性标记甲基赖氨酸和甲基组氨酸的化学方法，如 (单甲基赖氨酸Kme、二甲基赖氨酸Kme2、三甲基赖氨酸Kme3和甲基组氨酸HME) 与单分子荧光测序兼容。第二个创新是化学品的发展 低活性氨基酸的选择性标记方法，如酰胺(谷氨酰胺和天冬氨酸)、醚(蛋氨酸) 以及与单分子荧光测序兼容的烷烃(Ile、Leu、Val、Phe、Pro)。这些新的 单分子荧光测序的化学方法将导致氨基酸和PTMS的鉴定 具有高灵敏度、准确性和动态范围，能够识别低丰度的蛋白质和PTM 蛋白质组以高通量的方式覆盖整个范围。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过程，并导致不同的 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记物。这 研究还将使稀有蛋白质的检测成为可能，并可能发现新的分子调控网络 在细胞内，从而在基础科学和医学诊断方面打开了前所未有的机会。

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.