Study On The Proteomic Strategy Of Multi-dimensional Liquid Chromatography Coupling With Fast Digestion And Its Applications

With the accomplishment of the gene sequencing of Human Genome Project (HGP), proteomics research is presently one of the fastest developing areas of biological research in the post-genomic era. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. However, deciphering of a genome, the entire genetic information of an organism, is not equivalent to the actual understanding of it. Proteins represent the most functional component encoded for in the genome. In prince, the proteome, the total of protein expressed by a cell, a tissue or an organism at a given time and environment, can be predicted from the genome. The complex and dynamic nature of an organism's proteome, compared with the relatively static nature of the genome, makes this endeavor a complex task. These complexities include variations in mRNA editing, multiple protein processing pathways, and a wide array of co-translational and post-translational protein modifications that can affect the structure of a protein. This often results in the final protein product(s) differing from the predicted open reading frame encoded within a genome. Such gross and subtle modifications to a protein's amino acid scaffold can bring about dramatic changes in protein function, within the context of its intended biological activity. The task of identifying and characterizing these variations and modifications is one of the central challenges facing proteomic researchers today.Significant technological advances in proteomics approaches and instrument, as well as in related bioinformatics data analysis, have been achieved over the past decade. One of the most powerful of the proteomics approaches involves combing very high resolution and high efficiency separations with very high accuracy and high resolution mass spectrometry. 2-DE-MS is currently the most powerful approach in terms of overall separation power and sensitivity, and has been the primary focus of proteomics efforts at many labs. The large amount of data generated by utilizing high efficiency separations coupled with high mass accuracy mass spectrometry followed by data processing, analysis and display comprehensively characterize components within a complex protein mixture, including the ability to identify co-translational modifications, post-translational modifications, protein processing events, and discriminate between protein isoforms.2-DE is the most universal method for proteomics and posses high resolve power for proteins. Some problems in 2-DE, however, are still difficult to be overcome such as limited pH range, time-consuming, low dynamic range and inefficient sensitivity for low abundant proteins. The reality has motivated considerable effort in developing new technique to resolve proteomics as alternative approach to 2-DE.Strategies employed for characterizing proteomic samples generally follow either a "Top-Down" or "Bottom-Up" analytical methodology. Top-Down proteomic strategies rely on the separation and analysis of an intact molecule to discern information about protein function or identity. The classical example of this is gel electrophoretic techniques, where proteins are separated based on either the size and/or isoelectric point of the intact protein. Bottom-up methodologies differ from top-down methods in that they employ upfront proteolytic digestion of proteins. Subsequent mass analysis of the peptide digest mixture permits identification of proteins, and characterization of modification sites on those peptides. Although bottom-up strategies typically provide simple and direct means for protein identification, they are often less effective for characterizing modifications and protein variations, due to insufficient peptide coverage, dominant presence of the unmodified form, or the low abundance of the modified form of the protein. Combining information from top-down and bottom-up methodologies has existing precedent within the field of proteomics.Therefore, the core of this dissertation is presenting a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein separation (reversed phase liquid chromatography (RPLC) coupling with capillary isoelectric focusing (CIEF)) with on-line tryptic digestion followed by MALDI-TOF-TOF mass spectrometry. This dissertation is divided into four parts.In the first chapter, advances in 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. Combining information from top-down and bottom-up methodologies has existing precedent within the field of proteomics. The intention and meaning of this dissertation were explained.In chapter 2, we focused on the optimization of experimental conditions of on-target digestion of protein and its application. First, standard conditions for on-target tryptic digestion of proteins were chosen to be 1 ng of modified trypsin, high dynamic range for proteins, a 15-min reaction time and at the temperature of 50℃ in terms of speed and peptide sequence coverage using four standard proteins without any reduction or alkylation. Compared to long time in-solution digestion of thermal denatured proteins, on-target digestion shows higher efficiency due to much shorter time-reacting and higher peptide sequence coverage and produces similar protein identification patterns. The major advantage of the method is that it reduced the need for extra denaturation process and sample transfer, which improves the ability to detect low-abundance proteins (0.1-10ng/μl) and obtain good reproducible results. In addition, a very low amount of trypsin is required and few trypsin autolysis peaks are observed. Next, it is found that on-target limited digestion of protein can be controlled to obtain both PMF and MW of the protein, this can enhance the confidence of protein identification greatly, as demonstrated by the actual application of the method. Next, on-target multiple-enzyme digestion of protein is also investigated to raise confidence of protein identification, in particular, and post-modification proteins. Furthermore, we demonstrated that on-target low-volume digestion enabled capillary-LC separation with on-line MALDI target deposition and subsequent fraction-collection of the eluent using additional five model proteins, thereby lending itself to high-throughput identification of proteins. Finally, for the first time, the 2-D separation platform based on the liquid chromatography (SCX-RPLC) 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 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.In chapter 3, for the first time, a coupling of capillary reversed-phase liquid chromatography as the first dimension with capillary isoelectric focusing as the second dimension followed by on the MALDI target tryptic digestion for intact protein characterization was designed. But some important problems must be overcome. Based on two-dimensional separation system developed by our group, this work focused on incorporating tryptic digestion into the top-down proteomics methodology, retaining the benefits of the top-down method. First, an interface ofCIEF-MALDI is established to collect CIEF fractions into MALDI target effectively. Next, by constructing a hydrophobic layer of packing-material C18 coated with SE-30 on the MALDI target surface, we could permit the CIEF fractions to be easily concentrated and free of ampholytes using on-target washing. Following the removal of ampholytes, on the MALDI target tryptic digestion was performed to generate peptide mass fingerprinting for protein identification. Finally, based on the previous work, this part concentrates on the detection of protein MW and PMF, and the improvement of the removal of the carrier ampholytes for peptide mass fingerprinting (PMF) analysis. It is found for the first time that after on-target digestion of CIEF fractions, one-time washing using ammonium sulfate solution can remove ampholytes completely, and enhance the mass spectrum signal of digests, so the sample-handling procedure leads to great improvement of our experimental platform. It is also noticed that protein MW and PMF obtained by MS can enhance the confidence of protein identification.In chapter 4, an integrated protein separation/concentration/characterization platform, combining reversed phase liquid chromatography (RPLC) with capillary isoelectric focusing (CIEF) followed by MALDI-TOF mass spectrometry, has been constructed and developed further to provide not only significant protein resolution and high concentrating power, also physical properties of proteins for the accurate characterization of complex protein mixtures. First, using RPLC-CIEF-online digestion-MALDI-TOF-TOF-MS, we could obtain not only intact protein pI value, but also peptide mass fingerprinting for accurate protein identification. The feasibility of the strategy was first tested with a mixture of model proteins with different isoelectric points and molecular masses, and with rat liver extract further. A total of 376 proteins were identified. The results have shown that the proteome strategy is effective for fast characterization of proteins from complex proteomic mixtures. Finally, protein molecular weight is obtained from the mass spectrum and a 2-D plot of MW vs. pI analogous to 2-DE is generated. Comparison between theoretical and experimental intact protein MW values and pI values allows us to identify the proteins with similar physicochemical properties, including molecular weight (MW), isoelectric point (pI) and so on. Performance of the improved platform is demonstrated with rat liver tissue extracts. A total of 510 proteins were identified. The results have shown that the platform is very effective for complex proteomic analysis.In a word, the main contributes of this dissertation is the establishment and improvement of effective protein separation/concentration/characterization platform of RPLC-CIEF/on-line digestion/MALDI-TOF-TOF-MS. 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.