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: 10302032
• 负责人: Mingjie Dai
• 金额: $ 10万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302032
• 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 - 单分子蛋白识别和单细胞蛋白质组学 高通量DNA测序的最新进展已广泛改变了生物学研究和 生物医学，并导致单细胞测序和精确药物。与核酸相比，蛋白质更多 电流反映了细胞状态和动态变化，并被认为是更有效的生物标志物。 基于质谱的蛋白质组学患有有限的检测灵敏度（需要105-6个胡椒分子）， 并且不允许在小样品中有效检测低丰度的细胞蛋白和生物标志物（例如 单细胞或液体活检样品）。鉴于蛋白质扩增的PCR样自我复制策略是 不在视觉上，迫切需要开发一种无扩增的方法（即单分子）方法 准确，无偏的蛋白质识别和高通量分析。 该建议的目的是开发一种能够准确，高通量蛋白的新技术 来自单分子水平的未知样品的识别。这项研究的前提是超级 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 从单个完整的蛋白质分子中沿蛋白质的主要序列），该分子提供了准确的鉴定 和蛋白质分析的高吞吐量。该技术将结合高敏，高分辨率DNA- 涂料成像，高效蛋白质标记，蛋白质主链延伸和微流体对照 细胞操作。具体而言，我将开发两个目标：（1）开发生物化学，显微镜，生物物理学， 以及用于使用特定的高通量单分子蛋白鉴定的计算方法 氨基酸特征，（2）开发一个微流体工作流，完成单细胞裂解，蛋白质捕获和 修改和用于启用单细胞蛋白质组学的单分子成像。成功完成 拟议的研究将导致在广泛的基础研究和 临床环境，包括单细胞蛋白质组学（用于哺乳动物和细菌样品），发现低 - 抽象生物标志物和新病原体的鉴定。此外，新颖的概念和方法 在这项研究中开发的 未来生物物理研究和生物技术发展的基础。 这项K99/R00奖将促进我领导独立的学术研究计划的长期目标，并 开发智能分子工具，用于推进生物学研究和治疗疾病。培训 在K99期间提供的，在我出色的导师的指导下，将使我进一步扩展我的 科学知识和技术专长，以及发展我的专业技能以促进过渡 迈向独立的科学家和团队经理。