Phosphoproteome Analysis Based On Fluorescence Staining And Its Applications In Chang's And Nuero2A Cells

Phosphorylation and dephosphorylation of proteins are a ubiquitous and covalent modification in the living cells, and they are almost involved in all physiological and pathological processes, e.g., metabolisms, growth, development, carcinogenesis. Consequently, revealing the regularity of protein phosphorylation is crucial for comprehending the complexity of biological function of proteins. Because of their low stoichiometry and broad dynamic range, however, characterization of the phosphorylated proteins remains a considerable technical challenge. In the current study, A fluorescence staining method based on 2-DE was used to map phosphoproteins in bio-samples. When combined with total protein colouration methods, the approach can illustrate the phosphoprotein profile and total protein profile in one 2-DE gel. Moreover, by comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation can also be observed.In chapter one, we investigated the sensitivity, specificity and linear range of the Pro-Q Diamond staining method, and this method was validated in the qualitative and relative quantitative analysis of phosphoproteins.In chapter two, we applied the Pro-Q Diamond staining method for the detection of phosphoproteins in human liver Chang's cells, and establish an approach for systematic analysis of protein phosphorylation. A total of 269 phosphoproteins (including Group ) of Chang's cells was identified and 27 of them were validated phosphoproteins and embodied by the swissprot database. Simultaneously, we analyzed the phosphoprotein map on two-dimensional polyacrylamide gel and some guide line was found for the analysis of phosphoprotein in expression level, settling the base for the dynamic detection in vitro and relative quantification analysis of the phosphoprotein level.In chapter three, Pro-Q Diamond staining method was combined with DIGE (Difference Gel Electrophoresis) for relative quantitative analysis of phosphoproteins. Subsequently, we applied it to observe the temporal dimension of the DG/PKC signal pathway and obtained the global kinetic profile of phosphoproteins activation. 143 differentially expressed spots were found and 125 of them were identified with high confidence including 43 phosphoproteins embodied by PhosphoSite Database. Functional annotations for the identified proteins in Neuro2A cells were assigned according to gene ontology. The functions of annotated proteins are consistent with the cytological effects that PKC activation induced, which explained the molecular mechanism of DG/PKC signal transduction pathway in some extent.