| As the quest in understanding of life science advances, the demand for more powerful analytical tools becomes more and more urgent. Multi-dimensional separation technique becomes an effective solution for exploring complex system. Especially, the advancement of proteomics makes it essential to set up better analytical tools with higher performance, higher selectivity and higher peak capacity for separation and analysis of proteins and peptides. This dissertation focuses on coupling capillary isoelectric focusing (cIEF) with pressurized electrochromatography (pCEC) based on their orthogonal separation mechanisms. Samples can be separated by their isoelectric point, hydrophobic, molecular weight and charge condition in this system. It highlights on the key problems and relevant content in establishing both on-line and off-line cIEF/pCEC systems. The novel cIEF/pCEC two-dimensional (2D) system was realized for the first time and used in comprehensive analysis of complex peptides/proteins, demonstrating outstanding separation ability for complex biological sample and broad application prospects. The current 2D coupling system can be further extended to other multi-dimensional platforms, such as cIEF-LC, CE-pCEC, providing a strong technology fundamention for multi-dimensional separations and may promote the prosperous development of proteomics research.This dissertation is presented in five chapters, and the main contents are as following.The first chapter introduced the background of cIEF and pCEC from methodology, theory, detection method and applications respectively. Moreover, the outlook of multi-dimensional separation was summarized including MDLC, LC-CE and 2D-CE, where the development of cIEF or pCEC were outlined in detail. In chapter two, to establish a robust and high performance one-step cIEF, the major factors which affect the separation in cIEF were discussed, such as the coating method of the capillary, the concentration of carrier ampholytes, the focusing voltage as well as buffer additives. The HPC coated capillary was stable in 100 runs and proteins were separated better than in uncoated capillary. Besides, the reproducibility of the method was assessed by 6 standard proteins. A with-in day precision in RSD was less than 4% (n=5), while the between-day precision and bulk reproducibility were between 4-12%. Overall, this one-step cIEF, which was easy to operate, had a satisfied reproducibility and high focusing ability. It ensured an efficient separation of complex peptides/proteins in utilized condition.Separation conditions of peptides by pCEC were explored in the third chapter. The BSA tryptic digests were selected as the sample to evaluate the elution gradient, separation voltage, and reproducibility. With the increase of separation voltage, the separation velocity was increased. Compared to 0 kV, the resolution, column efficiency and capacity were all improved in -6 kV. The reproducibility was also satisfactory.Based on the research in two chapters above, chapter 4 focused on the key problems of establishing the off-line cIEF/pCEC two-dimensional system. The one-step cIEF which transferred samples by EOF made the two-dimensional process easier. It was much simpler and safer to collect the sample in the first dimension by introducing an electrical decoupler in the end of the capillary of cIEF. By a comprehensive separation of off-line cIEF/pCEC, more than 300 peaks were detected in BSA digests and the theoretic capacity was about 36000. Notablely, the capacity and separation ability were both significantly improved in comparison with a single dimensional method. Furthermore, this off-line 2D system was applied to the mapping of human red blood cell lysate (HRBCL) and its digests. A robust and high efficiency off-line cIEF/pCEC was set and laid a solid foundation for the on-line coupling.In chapter 5, we moved on to the construction of on-line cIEF/pCEC. To couple cIEF with pCEC on-line, the decoupler in the capillary of cIEF and a six port valve utilized as the interface. Meanwhile, HRBCL and its digests were respectively analyzed by this platform. Hundreds of compounds were detected by UV detector and detailed profiles were shown. In addition, cIEF/μHPLC, where no voltage was applied in the second dimension, can not match the column efficiency, separation velocity with on-line cIEF/pCEC. The overall separation ability of on-line cIEF/pCEC was much better, and it costs shorter time in comparison with off-line system. cIEF/pCEC 2D systems, including off-line and on-line, were established and evaluated for the first time. Both systems can separate complex peptides/proteins samples with high efficiency and capacity. The high focusing ability of cIEF and double separation mechanisms of pCEC play vital role in the successful junction. All these advantages make cIEF/pCEC system an effective and high performance technique in analyzing complex biological samples. The novel two-dimensional combination lays a solid foundation for the future study of multi-dimensional systems and may greatly affect the research in proteomics.
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