Single-molecule protein identification and single-cell proteomics

摘要-单分子蛋白质鉴定与单细胞蛋白质组学 高通量DNA测序的最新进展广泛地改变了生物学研究和 生物医学，并导致了单细胞测序和精确医学。与核酸相比，蛋白质更多 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。当前 基于质谱学的蛋白质组学存在检测灵敏度有限的问题(需要105-6个肽分子)， 并且不允许在小样本中有效地检测低丰度细胞蛋白和生物标记物(例如 单细胞或液体活检样本)。假设蛋白质扩增的类似于PCR的自我复制策略是 眼下还看不到，迫切需要开发一种无扩增(即单分子)的方法来检测 准确、无偏见的蛋白质识别和高通量分析。 这项提议的目标是开发一种能够准确、高通量地提取蛋白质的新技术 在单分子水平上从未知样品中进行鉴定。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 沿着蛋白质的初级序列)从单个完整的蛋白质分子中提取)，从而提供准确的识别 以及蛋白质图谱的高通量。这项技术将结合高灵敏度、高分辨率的DNA- 涂料成像、高效蛋白质标记、蛋白质骨架延伸和微流控 细胞操控。具体地说，我将发展两个目标：(1)发展生物化学、显微镜、生物物理学、 以及用于实现高通量、单分子蛋白质鉴定的计算方法 氨基酸特征，(2)开发包括单细胞裂解、蛋白质捕获和 修饰和实现单细胞蛋白质组学的单分子成像。圆满完成 拟议的研究将导致高通量、深入的蛋白质组研究在广泛的基础研究和 临床背景，包括单细胞蛋白质组学(针对哺乳动物和细菌样本)，发现低... 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 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 是未来生物物理学研究和生物技术发展的基础。 这一K99/R00奖项将有助于我实现领导独立学术研究项目的长期目标，并 开发用于推进生物研究和疾病治疗的智能分子工具。培训 在K99期间，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 一位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.