Advances in Large-Scale Bottom-Up Proteomics by Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Coupled with Prefractionation Steps

The objective of this dissertation is to make capillary zone electrophoresis - electrospray ionization - tandem mass spectrometry (CZE-ESI-MS/MS) a more useful tool in large-scale bottom-up proteomics.

CZE employs very simple instrumentation. Analytes are separated within a buffer-filled fused silica capillary under the influence of an electric field. However, traditional CZE has limited capability in analyzing large-scale complex proteomes.

To overcome this issue, I have worked to reduce sample complexity and enrich low abundance components while depleting high abundance ones. As a consequence, several prefractionation methods were employed before CZE-ESI-MS/MS to separate the entire proteome into simpler fractions. The number of protein and peptide IDs generated by CZE-ESI-MS/MS coupled with prefractionation steps did increase dramatically. The identification number produced by CZE-ESI-MS/MS can approach those produced by UPLC-MS/MS, but using nearly two orders of magnitude lower sample amounts. The quantification result generated by CZE-ESI-MS/MS was also evaluated.

With further improvements, including optimization of electrokinetically pumped sheath flow interfaces, improved sample preparation procedures, and reproducible separation capillary coating protocols, CZE-ESI-MS/MS will be a powerful alternative in large-scale bottom-up proteomics.