Development of high-sensitivity CE-ESI-MS-based methods for proteomic profiling of limited samples and single cells

Abstract

The mass spectrometry (MS)-based 'omics' approach to protein analysis enables high-throughput profiling of the proteome, providing a rich data set of protein identifications and abundances to probe global protein expression levels from cells, tissue, or other biological samples. The depth of proteome coverage is generally correlated with the amount of sample that is analyzed, which in many cases comprises microgram (µg) to milligram (mg) levels of starting protein amount for most informative coverage. The downside to high sample requirements is that the resulting measurements reflect a weighted average of protein levels from the bulk sample and lose any assessment of heterogeneity within the sample. Single-cell proteomics (SCP) can provide more detailed information about individual cells and reveal subtleties in cellular heterogeneity that have previously been masked by bulk sampling approaches. Achieving sufficient proteome coverage at the single-cell level to perform impactful biological research is a challenge that has fueled significant advances in all aspects of single-cell proteomic analysis. Additionally, for some types of samples, such as microbiopsies and rare cells, it is not feasible to obtain the amounts required for bulk analysis, making profiling of these mass-limited samples largely inaccessible without high-sensitivity proteomic techniques. This dissertation aims to exploit the strengths of capillary electrophoresis coupled to MS (CE-MS)-based technologies to develop innovative methods with improved sensitivity that can expand the capabilities of limited sample and single-cell proteomic analyses. Current state-of-the-art methods and strategies for limited sample and SCP, including sample processing techniques and high-sensitivity separations, are reviewed in Chapter 1. Chapters 2 and 3 explore the benefits of ultrasensitive CE-MS applied to bottom-up proteomic (BUP) analysis of low nanogram and sub-nanogram samples. First, a novel CE-MS-based bottom-up proteomics approach optimized for high sensitivity was described and compared with alternative ultrasensitive methods to highlight specific advantages for profiling post-translational modifications (PTMs) in limited samples. We hypothesized that ion mobility separation coupled with CE-MS would enable higher sensitivity in proteomic profiling. In Chapter 3, coupling ultrasensitive CE-MS with high-field asymmetric waveform mobility spectrometry (FAIMS) was investigated to achieve greater levels of proteome coverage from a low nanogram sample. For the analysis of intact proteins using the top-down proteomic (TDP) approach, we hypothesized that CE-MS-based methods present an ideal opportunity to inject intact cells and lyse directly in the separation capillary to minimize sample losses. The fourth chapter tests two different modes of cell injection for analysis of <10 mammalian cells and single cells with on-capillary lysis followed by CE-MS analysis exhibiting significantly higher numbers of protein and proteoform identifications from a single mammalian cell than have previously been reported. The results shown in this work denote a substantial step forward for the field of single-cell top-down proteomics. The final chapter summarizes the work detailed in this dissertation and discusses promising future directions for SCP technologies and prospective applications for CE-MS-based methods in other areas of high-sensitivity protein characterization. --Author's abstract