Study On The New Technique And Method Of High Throughput, High Sensitive And High Efficient Separation And Analysis For Proteomics Research

Scientific interest has been increasingly shifting to proteomics with theaccomplishment of the gene sequencing of Human Genome Project(HGP).Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. Theinvestigation of functional proteome is of great significance given that functions ofmost of genes are still not well understood. Presently proteomic research focuses ondifferential proteome analysis of human diseases, which is designed to find keyproteins with potentials to be used as markers for diagnosis or targets for medication.Proteomics is also becoming the spotlight of analytical chemistry. The fast development of proteomics relies on breakthroughs for high throughputanalytical technologies. 2D PAGE is the most universal method for proteomics andpossess high resolve power for proteins. Some problems in 2D PAGE, however, arestill difficult to be overcome such as limited pH range, time-consuming and inefficientsensitivity for low abundant proteins. The reality has motivated considerable effort indeveloping new technique to resolve proteomics as alternative approach to 2D PAGE. Liquid chromatography and electrophoresis are powerful technology to resolveproteins. But conventional one dimensional LC or CE are incapable of completelyresolving all the components in a complex mixture due to insufficient peak capacity.The development of two- or multi-dimensional column based separation techniquesgreatly improve the resolve power and peak capacity. During the past decade, a lot ofanalysts 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 part. In the first chapter, advances in 2D and multidimensional separation techniqueswere summarized with details in comprehensive 2D HPLC-HPLC, HPLC-CE andCE-CE system. Capillary array techniques and whole column image detector werealso discussed for high throughput analysis. In the second part of the dissertation, different aspects that can influence theperformance of CIEF were intensively studied and optimized. A primary study on a two-dimensional RPLC/CIEF separation with LIF detectorhas been described in the chapter 3. Capillary RPLC, the first dimension, providedhigh-resolution separations for proteins free of salts. CIEF, the second dimension, hadan orthogonal mechanism to cRPLC and provides excellent resolution capability for -X-复旦大学博士学位论文proteins with several orders concentration. Separation effectiveness of this approachwas demonstrated using complex protein/peptide samples, yeast cytosol and BSAtryptic digest. Apeak capacity of more than ten thousand had been achieved. A laserinduced fluorescence (LIF) detector developed for this system was used to achievehigh sensitive detection. A fmol level of peptide detection limit for BSA digest wasobserved. Two types of dyes, FITC and BODIPY maleimide, to tag the proteins werecompared. The thiol reactive dye was better fit for separations and detections of this2D system. In the chapter 4, based on array capillary isoelectric focusing (ACIEF),comprehensive two-dimensional system of cRPLC as the first dimension and ACIEFas the second dimension was developed for protein mapping. Combining the twoorthogonal high resolution techniques with parallel ACIEF separations, a highthroughput and powerful protein mapping is capable of being achieved. Laser inducedfluorescence (LIF) with whole column imaging detection (WCID) was designed inhouse for detecting protein bands in capillary array without mobilization afterfocusing. This imaging detection could avoid the possible problems likereproducibility, peak distortion, resolution loss arisen by mobilization. Such a systemof CRPLC-ACIEF-LIF-WCID, significantly enhancement of resolution, sensitivity,reproducibility and throughput was obtained. The efficiency o...