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 高通量DNA测序的最新进展广泛地改变了生物学研究， 生物医学，并导致了单细胞测序和精准医学。与核酸相比，蛋白质 直接反映细胞状态和动态变化，被认为是更有效的生物标志物。电流 基于质谱的蛋白质组学受到检测灵敏度有限（需要105-6个肽分子）的影响， 并且不允许有效检测小样品中的低丰度细胞蛋白和生物标志物（例如， 单细胞或液体活检样品）。考虑到用于蛋白质扩增的类似PCR的自我复制策略， 虽然还没有看到，但迫切需要开发一种无扩增（即单分子）方法， 准确、无偏见的蛋白质鉴定和高通量分析。 这项提案的目标是开发一种新技术，能够准确，高通量的蛋白质 在单分子水平上从未知样品中鉴定。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 沿着蛋白质的一级序列）与单个完整的蛋白质分子分离，这提供了准确的鉴定 和高通量的蛋白质分析。这项技术将结合联合收割机高灵敏度，高分辨率的DNA- PAINT成像、高效蛋白质标记、蛋白质骨架延伸和用于单克隆抗体的微流体控制 细胞操作具体来说，我将制定两个目标：（1）发展生物化学，显微镜，生物物理学， 以及使用特异性的核酸序列进行高通量、单分子蛋白质鉴定的计算方法 （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，用于实现单细胞蛋白质组学。成功完成 拟议的研究将导致高通量，深入的蛋白质组学研究在广泛的基础研究， 临床背景，包括单细胞蛋白质组学（用于哺乳动物和细菌样品），低- 丰度生物标志物和新病原体的鉴定。此外，新的概念和方法 在这项研究中开发的（例如，高效蛋白质-DNA标记，蛋白质骨架延伸）将形成 未来生物物理研究和生物技术发展的基础。 这个K99/R 00奖将促进我的长期目标，领导一个独立的学术研究计划， 开发智能分子工具，以推进生物学研究和疾病治疗。培训 在K99期间提供的，在我优秀的导师的指导下，将使我能够进一步扩展我的 科学知识和技术专长，以及发展我的专业技能，以促进过渡 成为一名独立的科学家和团队经理

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.