Developing and Optimizing a Robust Spray-Capillary Ce-Ms Platform for Ultrasensitive Omics Analysis

Single-cell multi-omics provides a vast amount of information, giving us an advantage in discovering mechanisms of gene regulation, protein expression dynamics, and epigenetic differences. This bolsters our understanding of cell heterogeneity in disease, which has implications for advancing diagnostics and treatments. Developing such methods may require the development of single-omics methods that could be paired. This thesis focuses on developing and optimizing an ultrasensitive, robust, and high-throughput spray-capillary CE-MS platform for multi-omics analysis of single cells.The spray-capillary CE-MS technology demonstrates high potential for emerging in single-cell and mass-limited sample analysis due to its enhanced detection limit and ultralow sample volume requirements. However, we had to address some challenges before proceeding with optimizing the system for mass-limited or single-cell analysis. These challenges included the rapid evaporation of such a small sample volume, in addition to the high fragility of the capillary’s tip. To mitigate these challenges, a humidity chamber was developed to maintain the small sample volume of a single cell, ensuring successful sample handling. Additionally, the tip structure of the capillary was modified to enhance the physical robustness of the tip and decrease the ESI voltage applied, which is expected to contribute to less oxidative interferences and a longer capillary life. The work also included optimizing the spray-capillary CE-MS system. First, we worked on improving reproducibility and robustness. Thus, a conditioning protocol was designed and further optimized for the top-down proteomics workflow. Second, to enhance separation and sensitivity, we optimized the CE separation voltage using the bottom-up proteomics workflow. These developments and optimizations were dedicated to enabling the analysis of mass-limited or single-cell samples by spray-capillary CE-MS. As a proof-of-principle experiment, HeLa protein digest single-cell samples were tested where peptides were detected from tiny fractions of single cells. Overall, this work has demonstrated that the optimized spray-capillary CE-MS platform implies a high potential for multi-omics analysis from single cells.