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

下一代单分子荧光测序技术2.0化学。

• 批准号: 10645898
• 负责人: Monika Raj
• 金额: $ 209.06万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645898
• 关键词: Affinity; Alkanes; Amides; Amino Acids; Basic Science; Biochemical Pathway; Biological; Biological Markers; Biological Process; Biology; Cells; Chemicals; Chemistry; Clinical; Collaborations; Complex; Complex Mixtures; DNA; DNA sequencing; Data; Detection; Development; Diagnostic; Disease; Early Diagnosis; Ethers; Genes; Genetic; Glutamine; Goals; Histidine; Human; Knowledge; Label; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Methylation; Modification; Molecular; Monitor; Mutation; Organism; Peptide Sequence Determination; Peptides; Periodicity; Positioning Attribute; Post-Translational Protein Processing; Posttranslational Amino Acid Modification; Process; Proteins; Proteome; Proteomics; Reading; Research; Research Proposals; Role; Side; Signal Transduction; Specificity; Speed; System; Techniques; Technology; Training; Variant; biomarker discovery; clinical diagnostics; gene product; genome sequencing; infancy; innovation; interest; new technology; next generation; novel; posttranscriptional; precision medicine; protein biomarkers; protein structure; single molecule; thioether; tool; transcriptome; transcriptome sequencing

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.

项目概要 人类蛋白质组极其复杂，包含 > 10,000 个蛋白质和每个蛋白质 100 倍的蛋白质形式 基因产物。在癌症和其他疾病中，突变、融合可能会产生一些新的蛋白质变体 以及进一步影响蛋白质功能和结构的 PTM。这需要识别 细胞或生物体中单分子水平的蛋白质和 PTM 以了解生物过程， 疾病分析和生物标志物发现。尽管蛋白质测序在彻底改变精度方面具有强大的力量 医学诊断方面，没有单分子方法可以在蛋白质组中鉴定蛋白质和 PTM。 宽层次。因此，对于蛋白质和 PTM 在生物学和生物学中的作用的理解存在巨大差距。 由于缺乏有效的技术来分析低丰度蛋白质和 PTM 造成的疾病 高度复杂的蛋白质组系统中的分子水平。本研究计划的主要目标是填补 在单点测序和鉴定蛋白质及 PTM 的可用技术范围内存在差距 分子水平。将开发一套新的化学方法用于侧链的特定修饰 氨基酸和 PTM 的反应性较低，因此难以修饰、附着各种荧光 肽的部分。作为一名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（Eric Anslyn 博士和 Ed Marcotte 博士），我们的定位 快速评估我们新开发的用于全蛋白质组分析的化学方法 吞吐量方式。新化学方法的高度化学特异性和产率将避免 单分子荧光测序对氨基酸的下游错误识别。拟议的研究 包含推进单分子蛋白质测序的各种创新。第一个创新，涉及 用于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 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。第二个创新是化学的发展 选择性标记反应性较低的氨基酸的方法，例如酰胺（Gln 和 Asn）、醚（Met） 和与单分子荧光测序兼容的烷烃（Ile、Leu、Val、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、准确性和动态范围，能够识别低丰度蛋白质和 PTM 以高通量方式进行蛋白质组范围的研究。因此，所提出的研究具有巨大的潜力 进一步我们了解这些 PTM 如何调节各种细胞信号传导过程并导致各种 疾病。这些工具将导致新型甲基赖氨酸和甲基组氨酸生物标志物的发现。这 研究还将能够检测稀有蛋白质，并可能发现新的分子调控网络 细胞内，从而为基础科学和医学诊断带来了前所未有的机遇。