| Proteomics has progressed enormously in recent years and been a favorite research means for post-genomics. It mainly refers to the followings: (1) Investigation of all the proteins expressed in a cell or tissue, which is named as expression proteome, and (2) study of protein expression changes in different stages of given samples by comparative proteomics, the functional proteome. This would allow the access to physiological/pathological mechanisms and key proteins with potentials to be used as markers. Proteomics has been widely applied for biological areas and become major route for effective discovering promising biomarkers despite of its new emergence. Currently, many international companies have invested greatly and the achievements in early diagnosis, targets discoveries, therapeutics judgements, and the discoveries and identifications of a number of disease or cancer-related proteins made proteomics study prospective.The main contributions of this dissertation are: establishment and optimization of 2-DE expression profiling of silkworm eggs, and differentially proteomic study on the sexual and parthenogenetical reproduction of silkworm. The dissertation consists of the following five chapters:In the first chapter, key techniques in the proteomics study such as 2-DE, MS, bioinformatics, and other related techniques have been reviewed. Applications of proteomics in silkworm species are summarized.The research in the second chapter centers on the obtaining of the samples for purpose of subsequently proteomics study. By using "warm water treatment" described by Astaurov, experimental parameters including different protection manners via manual egg-takeout, and various egg ages spontaneously laid after heat shock are estimated and optimized. The results show that heat shock could lead to satisfactory developmental eggs and hatch ratio of parthenogenesis, in which the incubate ratio is higher with the increased egg ages in limited range of times. Based on previously described work and present lab resources, enough samples required for subsequent experiments are obtained.The third chapter: A modified method of current rehydration loading in 2-DE procedure. In well-established 2-DE procedure, sample-loading step is crucial for 2-DE results, and rehydration loading and cup loading are the most commonly used methods up to present. However, due to adsorption of proteins to the chamber, rehydration loading results in up to 20-55% of overall losses of proteins loaded. Cup loading has been found to improve protein 2-DE patterns in comparison to rehydration loading. However, it is time-consuming and suffers from operational variability caused by manual manipulation between rehydration to IEF.In the experiment, a modified method is proposed with a multi-channel (8 channels) model of pipette as the applicator. The sample is loaded by "tapping" handling, in which rehydration buffer (RB) is introduced to accommodate the bottom of the strip holder first, sample solution is then added by "tapping" dropwise onto the surface of RB drops using the applicator. As a result, the contact of the sample with tray is kept at a minimum by the RB beneath the sample instead of mixing the two, so that proteins adsorption to the tray could be reduced. It is observed that the 2-DE performance is improved obviously compared to the rehydration loading. Also the use of multi-channel pipette could ensure the reproducibility of experiments. Furthermore, recovery of proteins from strips using the two different loading methods were investigated by the colorimetric assay, and the results show that proteins recovery of the modified method increased over 20% in comparison with that of rehydration loading. This verified the enhancement of proteins uptake by the proposed method.The four chapter: Development of an efficient approach for sample preparation. Undoubtedly, sample preparation is still the first and most important stage in 2-DE procedure. Due to the larger mounts of vitellogenin, egg-specific proteins (ESP) and No 30 family proteins (30KP) as major components in silkworm eggs, the 2-DE maps is less satisfactory because these abundant proteins prevent many biologically relevant spots from be detected. In this chapter, a new approach for sample preparation is successfully developed for 2-DE profiles of silkworm eggs. Though a peculiar way to lower the extent of abundant ESP and 30 KP in extracts, it is showed that spots on 2-DE were significantly enhanced (1268 ±24 versus 716 ± 19) after the unique pre-fractionation. Moreover, parts of proteins contributed from the novelapproach are excised for identification by MALDI-MS and MS/MS, and are classified bioinformatically respective to their function, biological process and cellular component. To our knowledge, by using the approach, so far the highest number of proteins in investigation of silkworm eggs is obtained.The approach introduced in this paper could offer more comprehensive information of 2-DE and thus make more spots to be visualized and identified. This would be a solid basis for either emphasizing on the establishment of proteins database or looking for quantitative changes in protein expression profiles in proteomic researches.In chapter five, comparative proteomics between the sexual and parthenogenetical reproduction of silkworms are performed. After the separation of the samples using 2-DE, 78 differentially expressed proteins are found. The proteins presented expressional change in the two different states are in-gel digested and further analyzed by MALDI-TOF-TOF MS, and overall 46 differentially expressed proteins are successfully identified. The database searches and characterization of the proteins identified suggest that these proteins might be mainly involved in such important processes as cell cycle, stress resistance, protein biosynthesis, transcription regulation and signal transduction. These proteins may function as different roles in parthenogenetic processes though diverse routes they participate in. The present work would deliver important data for better insights into mechanisms of parthenogenesis, and is of significance in finding out and mining of biological markers and proteins with biological importance.
|
PDF Full Text Request