Proteomic studies commonly utilize bottom-up proteomics due its high sensitivity, throughput, and robustness, bottom-up proteomics has issues with distinguishing proteoforms with high sequence similarity. Top-down proteomics overcomes this issue by analyzing intact proteins and identifying proteoforms and their post translational modifications’ (PTMs) with higher confidence providing opportunities to gain valuable insight into biological mechanisms. Reversed-phase liquid chromatography mass spectrometry (RPLC-MS) is the most widely used method for top-down analysis, there are still issues with sample loss facilitating a need to have micrograms of starting material. Capillary zone electrophoresis (CZE)-MS is a highly sensitive separation and detection technique that has emerged as an alternative to RPLC-MS for mass-limited samples, however applying CZE-MS for large-scale top-down proteomics has been impeded by the limited sample loading capacity and narrow separation window. This thesis will describe three projects improving CZE-ESI-MS for the large-scale top-down proteomics of complex samples.Chapter 2 and 3 focus on the improvement of single-shot CZE-MS/MS top-down proteomics. First, the systemic evaluation of the sample stacking technique, dynamic pH junction, for the focusing of proteoforms during CZE-MS. The optimized dynamic junction-based CZE-MS/MS platform reached 1-μL sample loading volume, 90-min separation window with high peak capacity (~280) for the identification and characterization of ~600 proteoforms from an E. coli proteome. The data in this work represents the largest loading capacity, separation window, peak capacity and proteomic identification of CZE for top-down proteomics of complex proteomes. In chapter 3, the length of the separation capillary was increased; the dynamic pH junction-based CZE-MS/MS using a 1.5-m separation platform achieved 180-min separation window with a 2-μL sample loading volume. This improved CZE-MS/MS platform produced high separation of myoglobin by baseline separating three proteoforms with over 100-fold concentration range and produced nearly 1,000,000 theoretical plates. The CZE-MS/MS platform also identified ~449 proteoforms from the E. coli sample using only 25 ng of proteins per run. Single-shot CZE-MS/MS identified over 1,500 and 2,000 proteoforms from two different regions of Zebrafish brain (cerebellum (Cb) and optic tectum (Teo)) when only 500-ng of material was loaded per run. Label-free top down proteomics of the two brain regions quantified thousands of proteoforms and revealed significant differences between the two regions. In chapter 4, we present for the first time a highly sensitive modified sample preparation workflow for top-down proteomics using laser capture microdissected (LCM) tissue samples of Zebrafish brain tissue. This workflow utilized OG, a MS-compatible detergent, while using a freeze/thaw method for protein extraction eliminating the necessity of detergent removal resulting in a lower sample loss for mass limited samples using top-down proteomics. This modified workflow identified an average of ~220 proteoforms of laser captured microdissected tissue sections (500-μm2 tissue section) when <250 cells were injected onto the capillary demonstrating the sensitivity of this platform for mass limited samples. This procedure facilitated quantitative top-down proteomics that produced protein expression profiles that can efficiently distinguish between different microdissected tissue sections even when the sample were isolated from the same brain region. This is the first attempt at utilizing LCM with CZE-ESI-MS/MS for highly sensitive top-down proteomics of Zebrafish brain tissues.
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