| Since 1950's, scientific interest has been increasingly shifting to molecular biology with the accomplishment of the gene sequencing of Human Genome Project (HGP). Proteomics has been the focus due to the role of executor of the life function. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. The investigation of functional proteome is of great significance given that functions of most of genes are still not well understood. Presently proteomic research focuses on differential proteome analysis of human diseases, which is designed to find key proteins with potentials to be used as markers for diagnosis or targets for medication. Proteomics is also becoming the spotlight of analytical chemistry.2D PAGE is the most universal method for proteomics and posses high resolve power for proteins. Some problems in 2D PAGE, however, are still difficult to be overcome such as limited pH range, time-consuming and inefficient sensitivity for low abundant proteins. The reality has motivated considerable effort in developing new technique to resolve proteomics as alternative approach to 2D PAGE.Liquid chromatography technologies are powerful technology to resolve proteins. But conventional one dimensional LC systems are incapable of completely resolving all the components in a complex mixture due to insufficient peak capacity. The development of two-or multi-dimensional column based separation techniques greatly improve the resolve power and peak capacity. During the past decade, a lot of analysts contributed to this field, such as Jorgenson's multi-dimensional separation, Yates' shot-gun technique, Aebersold's ICAT technique and so on.This dissertation is divided into 6 parts.In the first chapter, advances in 2D and multidimensional separation techniques were summarized with details. The theory basis of multidimensional separation techniques was introduced and the advantages and shortcomings of existing technical modes were discussed. The intention and meaning of this dissertation were explained.In chapter 2, we used rat as animal model to compare the effect of different extraction conditions on the extracted proteins. The extract methods of tissue sample at acidic, neutral and basic pH were described and compared by using two-dimensional gel electrophoresis and reversed-phase liquid chromatography. The images on gel and mass spectra showed that thedifferent method can enrich proteins at different pH range. Different pH extract methods can be applied into corresponding liquid chromatography mode with the compatible mobile phase so that the loss and precipitation could be avoided. The good reproducibility enables the sample preparation methods to be widely applied in proteomics analysis.In chapter 3, we describe an approach for fractionating complex protein samples from rat liver prior to 2-DE using RPLC. The study indicated that RPLC prefractionation can provide strong enrichment effect which enabled us to visualize additional and less abundance proteins. Chromatographic enrichment was also demonstrated by the peptide mass fingerprint data, which gave mass spectra with increased number of detected peptide and improved signal intensity. The reproducibility of this prefractionation technology allows pooling of several consecutive runs of the same sample, resulting in a highly enrichment of low abundance proteins. This technology route has been applied into the study of profiling mode of Chinese Human Liver Proteomics Project (CHLPP).In chapter 4, for the first time a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of immuno-affinity chromatography, was developed at the intact protein level for depletion of high abundance proteins from rat liver. The core idea was to deplete the high abundance proteins before the digestion.Proteins bound to the SCX phase were eluted off using a consecutive linear salt gradient. After salt fractionation, 62 fractions were obtained. Every fraction was separated further by RPLC. Ultraviolet signal was used to detect the separation process. The protein peaks with signal intensity above 0.1 AU was defined as high abundance proteins, whereas the other was defined as middle- or low high abundance protein. Then, the effluents contained high abundance proteins were collected individually and other effluents were pooled together. The combined middle- or low high abundance proteins were lyophilized, digested and separated by capillary reversed phase liquid chromatography (cRPLC). Mass spectrometry was used to analyze the protein. 62 fractions were treated using the same method. The results were that 77 high abundance proteins were depleted in an experimental process and identified proteins number was increased 2-3 times.Based on the results, a simplified rapid-speed and high-throughput protocol was put forward further. Instead of separating every fraction using cRPLC, all effluents containingmiddle- or low abundance protein were combined, enriched, digested and separated by online shotgun technology. The simplified method was used to analyze Chinese liver sample. 58 high abundance proteins were depleted and 1213 proteins were identified.In summery, the system presented a novel idea about the depletion of high abundance proteins. In contrast with affinity technology, this strategy with the advantages of low-cost and saving-time has no bias to any proteins and could realize the global proteins analysis.In chapter 5, for the first time, the 2-D separation platform based on the liquid chromatography combined with on-probe digestion technique and identification of mass spectrometry was constructed in intact proteins level for separation and identification of complex liver tissue proteins. The 2-D liquid chromatographic system was constructed by coupling SCX with cRPLC. Proteins were prefractionated by SCX according to charge in the first dimensional separation. 62 fractions eluting from the first dimension were subjected automatically to cRPLC to separate similarly charged proteins on the basis of their various hydrophobicities. A novel rapid on-target digestion technique was adopted to couple effectively the separation technique and mass spectrometry technique. After digestion, the matrix was deposited on the same spot, respectively. Then, the dried plate was analyzed by mass spectrometry. This new technology coupled the top-down approach and bottom-up technologies. The high efficiency of system was demonstrated for analysis of intact proteins from the soluble lysates of normal human liver. A total of 3313 proteins were identified.We further developed three-dimensional separation system combined SEC with the SCX/RPLC mentioned above. This 3D system exhibited the potential of further high resolution for extremely complicated mixture. The intention of 3D system was to realize the identification and quantification of proteins. We had explored some relative experiments and made some effective results.In a word, the main contributes of this dissertation is the development of effective separation system based multi-dimensional system. We aim at exploring and finding out new technological systems for proteomics, so that more breakthroughs can be obtained at the qualitative analysis and quantification study.
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