Evaluation of IMAC-based methods for enriching phosphoproteins
| Autor: | Tzung-Lin, Tsai, 蔡宗霖 |
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| Rok vydání: | 2007 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 In these years, as the rapid development of experimental techniques, scientists usually utilize proteomic methodologies, such as two-dimensional electrophoresis or mass spectrometry, to dissect the identity of differentially expressed proteins or the paths of signal transduction in the stimulated samples. Generally, such identification is generally fruitful when the target proteins are with high abundances. However, the target proteins with low abundance are sometimes hard to be isolated or identified. Unfortunately, even the protein phosphorylation account for the major regulations in cellular signal transduction, phosphorylated proteins are generally too few to be identified. Consequently, enrichment of phosphoproteins in biological samples is an extremely important task prior to the proteomic analysis. Currently, there are several approaches being utilized for purifying phosphoproteins. Among them, the immobilized metal affinity chromatography (IMAC), which was originally introduced by Lennart Andersson and Jerker Porath in 1986, is most utilized. The major advantages of using IMAC would be the low cost and the high capacity of absorbing phosphoproteins. Howerver, significant copurification of nonphosphoproteins is generally observed. There are many chromatographic factors, such as the type of charged metal ion, the type of chelating ligands, and the condition of system buffer may affect the result of IMAC based phosphoprotein purification. In this study, by systematically evaluating above chromatographic factors, we tried to elucidate the absorption mechanisms of phosphoproteins and nonphosphoproteins in the IMAC procedure. By utilizing a low molecular weight protein standard marker as evaluating model proteins, we have established two IMAC conditions efficiently enriching phosphoprotein from the model proteins. When utilized biological samples for evaluation, the phosphoproteomes from chicken egg white the human HepG2 cell lysates were effectively separated. However, applying a minor tuning to the original IMAC conditions was necessary for enriching the phosphoproteomes from the mouse myeloma SP2/0 and the rat kidney RMC cell lysates. With more understandings on the mechanism of IMAC based phosphoprotein purification, it is possible to optimize a feasible chromatographic condition for any selected materials. On the other hand, in conjunction with other proteomic techniques, the IMAC based phosphoprotein purification should provide a high throughput way for the study of phosphoproteomes. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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