Single-molecule protein identification and single-cell proteomics

单分子蛋白质鉴定和单细胞蛋白质组学

• 批准号: 10653329
• 负责人: Mingjie Dai
• 金额: $ 24.9万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653329
• 关键词: 2019-nCoV; Affinity; Aging; Amino Acids; Antibody Specificity; Award; Bar Codes; Basic Science; Binding; Biochemistry; Bioinformatics; Biological Markers; Biological Response Modifier Therapy; Biomedical Research; Biopsy Specimen; Biotechnology; Cell Culture Techniques; Cell Separation; Cells; Clinical; Complex; Complex Mixtures; Computer Analysis; Computing Methodologies; Cytolysis; DNA; Data Analyses; Detection; Development; Diagnostic; Disease; Future; Gene Expression Regulation; Goals; High-Throughput DNA Sequencing; Human; Image; Immobilization; Intelligence; Knowledge; Label; Lead; Liquid substance; Mass Spectrum Analysis; Mechanics; Membrane Proteins; Mentors; Methods; Microfluidics; Microscopy; Mitochondria; Modeling; Modification; Molecular; Nerve Degeneration; Nucleic Acids; Optics; Organelles; Outcome; Peptides; Preparation; Proteins; Proteome; Proteomics; Rare Diseases; Research; Resolution; Sampling; Scientist; Signal Transduction; Surface; Technical Expertise; Techniques; Technology; Therapeutic; Training; Vertebral column; Vision; base; biological research; biomarker identification; biophysical analysis; biophysical techniques; detection limit; detection method; detection sensitivity; emerging pathogen; innovation; liquid biopsy; miniaturize; molecular imaging; new technology; novel; precision medicine; prevent; programs; protein biomarkers; protein profiling; single cell sequencing; single molecule; skills; tool; tumor immunology

ABSTRACT – Single-molecule protein identification and single-cell proteomics Recent advances in high-throughput DNA sequencing has broadly transformed biological research and biomedicine, and led to single-cell sequencing and precision medicine. Compared to nucleic acids, proteins more directly reflect cellular states and dynamic changes, and are recognised as more effective biomarkers. Current mass spectrometry-based proteomics suffers from limited detection sensitivity (requiring 105-6 peptide molecules), and does not allow effective detection of low-abundance cellular proteins and biomarkers in small samples (e.g. single cells or liquid biopsy samples). Given that a PCR-like self-replication strategy for protein amplification is not within sight, there is an urgent need to develop an amplification-free (i.e. single-molecule) approach for accurate, unbiased protein identification and high-throughput profiling. The goal of this proposal is to develop a new technology that is capable of accurate, high-throughput protein identification from unknown samples at the single-molecule level. The premise of this research is that super- resolution microscopy can sensitively extract amino acid signatures (their abundances, or linear distribution along the protein’s primary sequence) from single, intact protein molecules, which provide accurate identification and high throughput for protein profiling. This technology will combine high-sensitivity, high-resolution DNA- PAINT imaging, high-efficiency protein labelling, protein backbone extension, and microfluidic control for single- cell manipulation. Specifically, I will develop two aims: (1) Develop the biochemistry, microscopy, biophysics, and computational methods for enabling high-throughput, single-molecule protein identification using specific amino acid signatures, (2) Develop a microfluidic workflow comprising single-cell lysis, protein capture and modification, and single-molecule imaging for enabling single-cell proteomics. Successful completion of the proposed research will lead to high-throughput, in-depth proteomic studies in a wide range of basic research and clinical contexts, including single-cell proteomics (for mammalian and bacterial samples), discovery of low- abundance biomarkers, and identification of new pathogens. Furthermore, novel concepts and methods developed during this research (e.g. high-efficiency protein-DNA labelling, protein backbone extension) will form the basis of future biophysical studies and biotechnological developments. This K99/R00 award will facilitate my long-term goal to lead an independent academic research program and develop intelligent molecular tools for advancing biological research and treatment of diseases. The training provided during the K99 period, under the guidance of my excellent mentors, will allow me to further extend my scientific knowledge and technical expertise, as well as to develop my professional skills to facilitate transition towards an independent scientist and team manager.

摘要 – 单分子蛋白质鉴定和单细胞蛋白质组学 高通量 DNA 测序的最新进展广泛改变了生物学研究和 生物医学，并导致了单细胞测序和精准医学，与核酸相比，更多的是蛋白质。 直接反映细胞状态和动态变化，被认为是当前更有效的生物标志物。 基于质谱的蛋白质组学检测灵敏度有限（需要 105-6 个肽分子）， 并且无法有效检测小样本中的低丰度细胞蛋白和生物标志物（例如， 假设用于蛋白质扩增的类似 PCR 的自我复制策略是 目前还看不到，迫切需要开发一种无扩增（即单分子）方法 准确、公正的蛋白质鉴定和高通量分析。 该提案的目标是开发一种能够准确、高通量蛋白质分析的新技术 在单分子水平上对未知样品进行鉴定是这项研究的前提。 分辨率显微镜可以灵敏地提取氨基酸特征（它们的丰度或线性分布） 沿着蛋白质的一级序列）来自单个完整的蛋白质分子，这提供了准确的识别 该技术将结合高灵敏度、高分辨率 DNA 分析和高通量。 PAINT 成像、高效蛋白质标记、蛋白质主链延伸和单通道微流体控制 具体来说，我将制定两个目标：（1）发展生物化学、显微镜学、生物物理学、 和计算方法，使用特定的方法实现高通量、单分子蛋白质鉴定 氨基酸特征，(2) 开发微流体工作流程，包括单细胞裂解、蛋白质捕获和 修饰和单分子成像，以实现单细胞蛋白质组学的成功完成。 拟议的研究将导致广泛的基础研究和领域的高通量、深入的蛋白质组学研究 临床背景，包括单细胞蛋白质组学（用于哺乳动物和细菌样本）、低 丰富的生物标志物以及新病原体的鉴定此外，新的概念和方法。 本研究期间开发的技术（例如高效蛋白质-DNA 标记、蛋白质主链延伸）将形成 未来生物物理学研究和生物技术发展的基础。 这个 K99/R00 奖项将促进我领导独立学术研究项目的长期目标 开发智能分子工具以推进生物研究和疾病治疗。 K99期间所提供的，在我优秀导师的指导下，将使我进一步拓展我的专业知识。 科学知识和技术专长，以及发展我的专业技能以促进过渡 成为一名独立科学家和团队经理。