NOVEL CE-MS-BASED APPROACHES FOR ULTRASENSITIVE HIGH-THROUGHPUT TOP-DOWN PROTEOMICS

Abstract

Proteins are essential biomolecules in living organisms, participating in a wide range of biological processes. Various forms of a protein, known as proteoforms, can originate from a single gene. These proteoforms arise through mechanisms such as genetic variation, alternative RNA splicing, and post-translational modifications (PTMs). It is estimated that approximately one million proteoforms are present in complex human biological samples. The study of proteoforms, or proteomics, is crucial for gaining a deeper understanding of the biological systems and their underlying molecular mechanisms. Researchers have recognized the unique advantages of analyzing mass-limited samples, such as clinical specimens and single-cell samples. This analytical approach demonstrates superior performance in applications including biomarker discovery, analysis of cellular heterogeneity, and investigations into drug mechanisms. Two main strategies have been developed to address proteomic analysis: top-down proteomics and bottom-up proteomics. Unlike the bottom-up approach, top-down proteomics does not require digestion of intact proteins into peptides. As a result, it enables more accurate characterization of biological features such as post-translational modifications (PTMs), offering a distinct advantage in preserving protein-level information. However, due to the complexity of sample, wide dynamic range of proteoform abundant, and low signal intensity, top-down proteomics are challenged by low sensitivity and low proteome coverage. The technic and method development for top-down proteomics are essential. Previously, our group developed a novel sample injection device known as the spray-capillary. By utilizing the pressure difference generated at the tip during the electrospray ionization (ESI) process, this device enables robust and quantitative sample injection at ultralow flow rates—as low as picoliters per second (pL/s). The spray-capillary can also be integrated into capillary electrophoresis (CE) systems, allowing ultra low volume sample injection coupling with online CE-MS analysis. This platform has demonstrated its applicability in single-cell metabolomics and automated, high-throughput bottom-up proteomics. The present dissertation focuses on the optimization and application of the spray-capillary-based CE-MS platform for top-down proteomics. The first project focused on optimizing the spray-capillary CE-MS platform for top-down proteomics, including the evaluation of capillary coatings and inner diameters. Using this optimized setup, we successfully demonstrated the analysis of low cell number samples. The second project, presented in Chapter 3, involved adapting a multisegment sample injection strategy to our platform to enhance analytical throughput. Following configuration optimization, the throughput was improved by approximately ten-fold. The final project addressed challenges in quantification associated with MS1-based methods from the second study. To improve quantification accuracy and sensitivity in multisegment sample injection analyses, parallel reaction monitoring (PRM) was implemented, resulting in significantly enhanced quantitative performance.