| The completion of the human genome project has facilitated the entry of biomedical research into the post-genome era-proteomics research. There are two technical routes in proteomics:peptide-centric bottom-up and intact-protein level top-down approaches."Top-down" proteomics can provide more information for intact proteins which can be used to study protein quantitation, protein-protein interactions (PPIs) and posttranslational modifications (PTMs). The strategies based on separation and identification of intact proteins has become the mainstream in proteomic research. However, proteins are more complex than peptides. The efficient and high throughput separation of complex protein mixtures at intact-protein level is a great challenge to the chromatographic field. Construction of high-throughput multi-dimensional array liquid chromatography platform has great potential for large-scale proteomics analysis.The elucidation of PPIs is one of the most challenging and important tasks in proteomics research. Study of PPIs can provide important information for understanding protein functions, elucidating disease pathogenesis and developing new drugs. So PPIs analysis has become the focus of proteomics. The current technologies have hindered high throughput screening of PPIs because they are usually time-consuming and expensive. Therefore, it is urgent to develop new strategies for the large-scale study of PPIs.We focus on the key and challenging problems in proteome field, a novel kind of intact-protein trapping columns was developed for trapping and desalting of proteins. We established a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for study of PPIs was proposed. Differential proteomics of normal and fat rat liver proteins was achieved based on label-free proteomics. The research work in this thesis is divided into five chapters.In Chapter1, advance and application of monolithic columns were included, development and application of multidimensional liquid chromatography in the field of proteome were introduced, technologies for analyzing PPIs were summarized. The research background was demonstrated. In Chapter2, most commercially available precolumns and our previously developed precolumns are mainly employed for trapping peptides, it is urgent to develop intact-protein trapping columns for high efficient trapping and elution of proteins. A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol-gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to300bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8monolithic trapping columns was99.3%on average. The loading capacity of5mm×320μm i.d. C8trapping columns for the protein mixtures was30μg. The C8trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.In Chapter3, we established a capillary two-dimensional array liquid chromatography system for automated, high-throughput analysis of intact proteins, in which one strong-anion-exchange (SAX) chromatographic column was used as the first separation dimension and18parallel capillary reverse-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Proteins eluting from the SAX were trapped and desalted by18parallel intact-protein trapping columns. Then protein fractions were back-flushed simultaneously from18intact-protein trapping columns to capillary RPLC columns. The eighteenplexed capillary array chromatography system is capable of concurrently separating eighteen different samples, resulting in an18-fold increase in analytical throughput. Protein fractions eluting from RPLC columns were spotted onto the MALDI sample plate in1min intervals through an array of capillary tips. On-plate tryptic digestion technique was employed to digest the effluents. Normal mouse liver tissue proteins were analyzed by the fully multiplexed high-throughput two-dimensional liquid chromatography platform coupled by MALDI-TOF-TOF-MS identification. In total,1030proteins were identified, which proved the system’s promising potential for intact-protein separation in proteomics.In Chapter4, we proposed a new method for PPIs research. Bait protein was immobilized on amino particles, prey protein was labeled by a fluorescent reagent, and then they were incubated, eluted, detected by fluorescence microscopy. GST and its antibody were used to verify the feasibility of the method. First, amino-nano-magnetic microspheres were synthesized, glutaraldehyde was connected with amino of magnetic particles by chemical reaction, and then protein was connected to glutaraldehyde. After protein immobilization procedure was conducted, the UV absorption value of the supernatant solution was measured at280nm to calculate the amount of protein immobilized on the magnetic microspheres. A solid-support reaction was described to realize fluorescent derivatization of proteins. A simple, low-cost homemade capillary C18cartridge was fabricated as the solid-support reactor, protein was captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. By incubating immobilized protein and FITC-labeled protein in Tris-HCl buffer (pH7.5,100mM NaCl,4℃), we investigated PPIs. This new method was applied to study PPIs between human serum albumin (HSA) and human plasma fractions.In Chapter5, we reported on differential proteomics of normal and fat rat liver proteins. This was achieved by2D-LC-MS/MS-based label-free protein quantification. The peptides were desalted, dried and then resuspended with60μL volume of loading buffer (5mM Ammonium formate containing5%acetonitrile, pH3.0), separated and analyzed by2D strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (2D-nanoLC/MS). The experiments were performed on a Nano Aquity UPLC system connected to an LTQ Orbitrap XL mass spectrometer equipped with an online nano-electrospray ion source. A20μL plug was injected each time to form the salt step gradients. Ten step gradients were carried out. The plugs were loaded onto the SCX column with a loading buffer at a20μL/min flow rate for5min. A19μL peptide sample was loaded onto the SCX column before the gradient plugs were injected. The eluted peptides were captured by a Captrap Peptide column, while salts were diverted to waste. To recover hydrophobic peptides still retained on the SCX column after a conventional salt step gradient, a RP step gradient from15%to50%acetonitrile was applied to the SCX column. After all MS/MS spectrums were identified, we found quantitative differences of31proteins common to two samples, of which,21up-regulated and10down-regulated.In summary, this thesis focuses on the key and challenging problems of proteomics. We developed a novel type of intact-protein trapping columns for desalting and trapping of intact-protein. We set up a multidimensional capillary array liquid chromatography platform coupled by MALDI-TOF-TOF identification of intact proteins. And a new method for studying PPIs was proposed. |
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