Method development for capillary electrophoresis mass spectrometry (CE-MS)-based proteomics and application to uncovering proteome dynamics of zebrafish embryos during early embryogenesis

Reversed-phase liquid chromatography (RPLC) coupling with tandem MS (MS/MS) is often the method of choice in both peptide-centric bottom-up proteomics (BUP) and proteoform-centric top-down proteomics (TDP) studies. In recent years, capillary zone electrophoresis (CZE)-MS has attracted attention as another platform in proteomics due to high separation efficiency, high sensitivity, and complementarity to LC-MS. This work is dedicated to developing novel CE-MS-based methods for large-scale proteomics and applies them to study the proteome dynamics of zebrafish embryos during early embryogenesis.In Chapter 2, a sample stacking method, dynamic pH junction, was systematically investigated and employed to improve CZE’s sample loading capacity for large-scale BUP. The results of the optimized system represent the highest loading capacity, the highest peak capacity, and the widest separation window of CZE for peptide separation to date. The automated CZE-MS system opened the door to using CZE-MS for large-scale BUP. In Chapter 3, for the first time, a strong cation exchange (SCX)-RPLC-CZE-MS/MS platform was established for deep BUP and phosphoproteomics. The platform approached comparable performance to the modern 2D-LC-MS/MS for deep proteomic sequencing evident by identifying 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest, 11,555 phosphopeptides from the HCT116 cell line. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein, peptide, and phosphopeptide IDs. In Chapter 4, a quantitative BUP study was performed on zebrafish embryos across four developmental stages during the maternal-to-zygotic transition (MZT) via coupling isobaric tag for relative and absolute quantitation (iTRAQ) chemistry with both RPLC-MS/MS and CZE-MS/MS. Expression kinetics of nearly 5000 proteins including over 100 transcription factors (TFs) across four early embryonic stages were determined. The protein expression profiles fall into several different clusters and accurately reflect the important events during early embryogenesis. Further studies of the expression profiles of TFs revealed that the differentially expressed TFs during the MZT show wave-like expression patterns. Top-down proteomics (TDP) aims to directly characterize proteoforms in cells. CZE-MS/MS has been demonstrated as a useful tool for TDP. In Chapter 5, for the first time, we evaluated various semiempirical models for predicting proteoforms’ electrophoretic mobility using large-scale TDP data sets from earlier CZE–MS/MS studies. Linear correlations were achieved between the experimental and predicted μef of E. coli proteoforms and histone proteoforms (R2 = 0.98), demonstrating that the μef of proteoforms in CZE-MS can be predicted accurately, which could be useful for validating the confidence of proteoform IDs from a database search. In Chapter 6, we concluded the results of this dissertation and provided our expectations for future studies.