New Technologies Of Bio-mass Spectrometry And Their Applications In Disease Proteomics

The main contributions of this dissertation are: a modified in-gel digestion protocol was developed; a new online preconcentration approach was developed; a series of new mesoporus materials were investigated for their potential as MALDI matrices; and differential proteomic analysis was carried out for ischemic heart mitochondrion and liver carcinomas with high and low potential for metastasis. Scientific interest has been increasingly shifting to proteomics with fast measurement of genomic sequences. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. The investigation of functional proteome is of great significance given that functions of most of genes are still not well understood. Presently proteomic research focuses on differential proteome analysis of human diseases, which is designed to find key proteins with potentials to be used as markers for diagnosis or targets for meditation. Proteomics is also becoming the spotlight of analytical chemistry. The fast development of proteomics relies on breakthroughs for high throughput analytical technologies which include two-dimensional electrophoresis, biological mass spectrometry and bioinformatics. This dissertation consists of two parts. In the first part, a proteomic platform based on two-dimensional electrophoresis and mass spectrometry was established. With this platform, in-gel digestion methodology was investigated and a modified in-gel digestion protocol and a new online preconcentration approach were developed. Also a preliminary investigation was carried out for inorganic MALDI matrix. In the second part, differential proteomic analysis was carried out for ischemic heart mitochondrion and liver carcinomas with high and low potential for metastasis.Part I: Establishment and development of proteomic platform.Polyacrylamide gel electrophoresis, either in one or two dimensions, is the most commonly used technique for separation of complex protein samples. And in-gel digestion combined with mass spectrometry has become a major route for protein identification. Various versions of protocols for tryptic in-gel digestion of CBB-stained proteins have been documented and all of them require a thorough removal of the dye with extensive washing steps prior to proteolysis. The washingsteps slow down the procedure and cause sample loss. Accordingly a simplified protocol was proposed which does not demand a removal of the dye. The simplified protocol was tested using 500 ng BSA. The result of HPLC/ESIMS showed tryptic digestion was hardly affected by CBB. Because CBB is strongly retained in RPLC, it does not affect the analysis of peptides in MS. A comparison between the simplified protocol and the conventional one revealed that the simplified protocol tended to produce more incompletely cleaved peptides, which is favorable for improvement of sequence coverage. Experiments with 50 ng BSA showed that the simplified protocol can be used for MALDI-TOFMS with sufficient sensitivity. The simplified protocol was further applied to the analysis of two protein spots on a 2DE map of rat heart mitochondrion.A new method was developed for on-line pre-concentration of peptides from protein digested in-gel prior to HPLC-ESI-MS-MS analysis using strong cation exchange (SCX) chromatography. Peptides were directed loaded onto a capillary SCX column without any pretreatment, and then displaced using 1 M ammonia acetate to a reversed phase LC-MS system. A 30 fold concentration was achieved accordingly. The method was validated with tryptic peptides of bovine serum albumin (BSA), in which 100 ng of BSA in polyacrylamide gel was successfully identified. Compared with the conventional technique, more peptides were detected with less sample loss and the ability of removing contaminants by SCX chromatography. Consequently, the new approach offers less time consuming, easier to automate, and more favorable for LC column protection and analysis reproducibility.The introduction of organic matrices to MALDI-MS greatly advanced bimolecul...