Study Of The New Proteomic Quantification Strategy Combined With Fluorescent Labeling And Multi-Dimensional Liquid Chromatography

Biology in general and systems biology in particular increasingly requires the detection and quantification of large numbers of analytes. Since only the quantity of proteins can reflect the status and changes of a biological system, quantitative proteomic nowadays plays an important role in the comparison of biological systems. For the past decades, different techniques for this purpose have been developed. In this study, we focused on developing novel quantitative proteomics strategies based on multi-dimensional liquid chromatography, inventing OPA and IAF fluorescent labeling method for proteins quantification and establishing a new protein quantification strategy based on N-terminal peptide.In chapter1, the recent developments of the separation and identification techniques for proteomics analysis were reviewed. The requirements and current techniques for multi-dimensional liquid chromatography separation were introduced. The research background was demonstrated.In chapter2, an easily replaceable microchip enzymatic micro reactor has been fabricated based on the glass microchip with trypsin-immobilized superparamagnetic nanoparticles. Amine-functionalized magnetic nanoparticles with trypsin immobilized were packed onto the glass microchip by the application of a strong magnetic field. Complete protein digestion was achieved in a short time (10s) under the flow rate of5μL/min. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. It takes less than1min under the condition of extra magnetic to form a new packing bed. This micro reactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. Besides, an on-line sample enrichment system was evaluated and applied to trace-level bio-molecules quantification using monolithic trapping columns in capillary liquid chromatography (LC) system. The monolithic trapping column is used in front of the analytical column to increase sensitivity, enhance sample loading amount and minimize the loading time. Online trapping system has the advantages of easy handling and labor saving by the column switch system. Results demonstrated high efficiency of the monolithic trapping columns for trapping analytes in online capillary LC system. In chapter3, a simple OPA method of solid-support labeling for picomoles of proteins in diluted solution was developed. A C18cartridge was used for proteins capture and subsequent labeling with OPA. The use of the solid-support reactor allows easy handling for protein enrichment and purification. The employed OPA reagents for protein assay give no self-fluorescence. The application of OPA labeling method leaved no potential interferences for the assay and extra separation step of the proteins from interferences. Using Casein as a test protein, we have labeled and detected5ng/uL casein. This method was applied to analyze the fraction from weak anion-exchange chromatography (WAX) of human liver extract.In chapter4, a facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using mass spectrometry (MS) for quantification, the FLAQ method is a chromatography-based quantification in combining with MS for identification. Multi-dimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. A fluorescence dye,5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALD1MS identification. The limit of quantitation for the model protein was as low as0.34amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.In chapter5, a new strategy for protein quantification was studied based on N-terminal peptide of proteins. In detail.(1) Based on N-peptide isolation, quantification information of proteins can be obtained by HPLC separation.(2) Using the functional materials and amino group blocking reactions, the terminal peptides were successfully separated and isolated.(3) Qualification and identification of proteins by the technology of N-terminal peptide location. By the new technology, protein can be quantified with high accuracy and reliability. By the development of novel technology for protein quantification, we are promising to supply the techniques for the protein science to find potential functional proteins and biomarker.