New Techniques And Methods In Multi-dimensional Chromatography-mass Spectrometry Analysis Platform For Proteomics Research

After the accomplishment of human genome sequencing, the proteome research has been initiated. Proteomics constitutes of expressional proteomics and functional proteomics. Expressional proteomics includes qualitative and quantitative study of proteins expressed in cells, the study of proteins cellular location and post-modification. Functional proteomics focused on the researches of protein interactions, functions played by proteins in specific pathways and cellular structures, and relationships between proteins structures and functions. It is necessary to perform a sufficient separation of proteins prior to identify them because proteins expressed in cells are very complicated, generally tens of thousands proteins are expressed in eukaryotic cells. 2-D Gel combines IEF separating proteins according to pI and SDS-PAGE separating proteins according to MW. It is the most widely used technique in protein separation since its establishment in 1975, however it has difficulties in separating hydrophobic proteins or proteins with extreme mass. Its dynamic range is only 4 orders of magnitude. Also it is labor-intensive and time-consuming and can not be hyphenated with mass spectrometer in an automated way. All these disadvantages limit its wider utilization. The Faster, more efficient and highly automated separation and analysis platforms have to be developed for proteomics research.Recently, one-dimensional high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been used for protein and peptide separation, but their resolution ability and peak capacity are limited. To achieve higher separation ability, multi-dimensional separation system has been established by hyphenating chromatography and electrophoresis. Compared to one-dimensional separation, multi-dimensional separation features largely in higher peak capacity and better resolution. According to the mathematics model developed by Giddings, the peak capacity of multi-dimensional system is the product of peak capacity in each component mode. A higher peak capacity is beneficial to complex sample separation. To establish a multi-dimensional separation platform, there are mainly two factors to be considered carefully. One is how to choose appropriate separation modes for hyphenation and the other is how to hyphenate them. For the first issue, the modes with orthogonal separation mechanisms should be combined for maximal peak capacity; also the compatibility of mobile phases in different modes should be givenconsideration to. For the second issue, different devices, such as variant interfaces, loops or trapcolumns, have been utilized to realize hyphenation. The hyphenation can be performed between HPLC and HPLC, CE and CE, or even HPLC and CE. Researchers can choose component separation mode based on sample property and the practical experimental conditions.After separation, protein or peptide fractions are introduced into mass spectrometer by ionization, then performed with MS/MS analysis and the resulting data are searched against protein sequence database for identification. To develop novel multi-dimensional separation platforms and hyphenate them with bio-mass for complex bio-sample analysis, especially for expressed proteome analysis in mammalian tissues or cells, is the theme of this dissertation.Chapter 1 reviews the advances in multi-dimensional separation techniques, especially focuses on the establishment and application of different two-dimensional and three-dimensional separation systems in proteome research.Chapter 2 focused on the optimization of experimental conditions in on-line 2-D LC-MS/MS system and its application. We have determined the LOD of this system and evaluated its performance by analyzing a test sample containing 18 standard proteins. The stepwise salts elution gradient in SCX and continuous elution gradient in RPLC have been optimized. We also compared the commercial trapcolumn and the home-made large-bore particle-entrapped monolithic precolumns in terms of the ability of trapping and concentrating peptides, and results showed that the home-made precolumns can take place of the commercial ones for real sample analysis. The nano-flow 2-D LC-MS/MS system has been used for analysis of sequentially extracted protein sample from LCI-D20 mouse liver. There were 450 soluble proteins, 339 urea-solublized proteins and a total of 687 proteins identified. C57 mouse liver nuclear proteome was also studied by this on-line 2-D system and a total of 462 proteins identified from 5 identification processes.The development of parallel MS/MS analysis strategy and its combination with 2-D LC for complex protein sample analysis was described in Chapter 3. The ionization mechanisms of ESI and MALDI are fundamentally different, which makes the information of the same sample obtained by the two approaches complementary and analogous. The parallel MS/MS analysis strategy took advantage of complementary nature of ESI and MALDI and combined them to glean morecomprehensive information from one sample. By using a splitter, we realized the hyphenation of 2-D LC system with ESI-MS/MS and MALDI-MS/MS simultaneously. The effluents of RPLC were split into two parts, one part was introduced into nano-ESI and the other part was deposited on MALDI target plates for MALDI-MS/MS analysis. This parallel analysis strategy was used for normal human liver expressed proteome analysis and the identification results demonstrated the superiority of parallel MS/MS analysis over the single MS/MS analysis in terms of identifiable protein number and identification confidence.In Chapter 4, a work of coupling comprehensive two-dimensional capillary liquid chromatography and capillary electrophoresis system with tandem MALDI-TOF-TOF-MS was first reported. Two novel interfaces, i.e. valve-free hydrodynamic sampling interface and CE-MALDI interface, were designed and constructed to connect RPLC, CZE and MALDI-MS. The novel cRPLC-CZE interface enabled a positive high voltage applied on the ends of CE capillary, which facilitated the combination of CE with mass spectrometer. The CZE-MALDI interface was used to realize the stable deposition of CE effluents on MALDI target plates during CE separation. We also optimized the experimental conditions of this 2-D system, including the operation of RPLC-CZE interface, CE separation voltage, the selection of CE running buffer and its concentration, the frequency of sample spotting. The total separation time of the RPLC-CZE system was equal to the time needed by one dimensional RPLC separation and its peak capacity was increased by at least one order of magnitude. The efficiency of the overall system was evaluated by analysis of liver proteins in HCC (Hepatocellular carcinoma) nude mice model with high metastasis potential (LCI-D20). A total of 1350 MS/MS spectra were generated, more than 2000 peptides with S/N above 20 and 384 proteins were identified after about 90min separation, which demonstrated the application potential of this system for fast and high-throughput proteomics research.In chapter 5, three-dimensional SEC-SCX-RPLC-ESI-MS/MS system was developed, which was based on the 2-D LC system established in chapter 2. This 3-D system aims to provide more sufficient separation and more confident identification by introducing an additional separation prior to 2-D LC system. Size exclusion chromatography (SEC) was selected as the new separation dimension for its non-bias effect on sample, multiple choices of mobile phases ranging from acidic aqueous solution to basic solution, even organic solvents.The total peak capacity of 3-D LC was greatly improved because of the additional separation. The sample capacity of the three dimensional system was also increased greatly due to the large I.D. column used in SEC.For real sample analysis, there are two types of prefractionation methods by using the 3-D system, one is performed at protein level, after SEC separation, fraction collection was performed manually, and each fraction was terminated when a significant decrease in absorbance was observed in the UV spectra, then lyophilized the fractions and digested them for the following 2-D LC-ESI-MS/MS analysis. The prefractionation could also be performed at peptide level. First, the extracted proteins were digested and fractionated by SEC, and then each peptide fractions were lyophilized, redissolved and analyzed by 2-D LC-ESI-MS/MS. We have compared 2-D LC, 3-D LC with protein prefractionation and peptide prefractionation for complex protein sample analysis in terms of identifiable protein number and identification confidence. The 3-D LC employing protein level prefractionation was more effective than the other two approaches. The three-dimensional SEC-SCX-RPLC-MS/MS system has been utilized for analyzing proteins expressed in normal human liver tissue and a total of 1624 proteins were identified at high confidence level (>95%). According to bioinfonnatics analysis of the identification result, these proteins located in different subcellular locations, played diverse molecular functions and participated in different biological processes, which demonstrated the powerful identification ability of this 3-D LC-MS/MS.