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 ...