| Protein phosphorylation is one of the most important post-translational modifications in cells, and the reversible phosphorylation of proteins regulates nearly every aspect of cell life from signaling transduction, metabolism, gene's expression to cell growth, division, differentiation and development. Moreover, the disregulation of protein phosphorylation will induce many human diseases, most notable cancer, diabetes, heart disease, Alzheimer's disease, and so on. So phosphoproteomics has been a hotspot in proteomics research and detecting phosphoproteins and defining their phosphorylation sites are very important to understand the mechanism by which phosphorylated protein affects a biological pathway. However, because the phosphoprotein content is generally low, analysis of phosphoproteins is not straightforward and a large-scale analysis of phosphoproteins in a cell or tissue is still a big technical challenge in phosphoproteomic research. Neural stem cell performs many complex and essential functions, which are most involved reversible phosphorylation regulation. The phosphoproteome analysis of the neural stem cell allows us to further understand and recognize the complex function and the essential regulation of phosphoprotein in division, growth and development of neural stem cells.Our research contain following three parts:1) Developing methods for protein phosphorylation analysis based on the combination of a new labeling regeant to phosphor group and the related mass spectrometry; 2) Constructing the data analysis platform for the large scale phosphoproteomics; 3) Analyzing the phosphoproteomics of proteins from neural stem cells.In the first part, a systematic analysis methods for phosphoproteins has been established. The standard protein digest mixtures were converted to the corresponding methyl esters (that is, CH30-) at first. Then the methylated peptides were subjected to a one-pot reaction in the presence of carbodiimide (that is, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDC), imidazole and a reagent (MPAE,2-(4-MethylPiperazin-1-Aacetamido)Ethanaminium) which was synthesized in this lab and was designated to label and indicate phosphate groups in the process of MS analysis. When MS/MS analysis, the phosphopeptides which are labeled by MPAE will disrupt the carboxyamide bonds and form a report ion, which is 113 in the "quiet region" of the MS/MS spectrum. Thus we can differentiate the phosphopeptides and non-phosphopeptides by the report ion(113 in the MS/MS spectrum). Based on the method, we can identify all the phosphopeptides and non-phosphopeptides in one LC/MS-MS run.In the second part, the data analysis platform for the large scale phosphoproteomics is established. The database search parameters are optimized to make sure that the result is precise and reliable. It can lay the foundation for the large scale, high-throughput phosphoproteomics analysis. A phosphorylation site location method and procedure for our experiment has been developed, and it is convenient for the phosphorylation site analysis of proteins from neural stem cell line.In the third part, a robust and automatic system was developed, and phosphoproteomics analysis of proteins from neural stem cell line(C17.2 cell line) are reported here. A total of 259 phosphoproteins,337 phosphopeptide sequences are determined,407 phosphorylation sites are defined, and 18 of those sites are newly identified. These results would allow further insight into the mechanism of stem cell's division and growth and may help to search some biomarkers for neural tumor.In total, a noval system strategy was developed for phosphoproteomics. These methods in our strategy can be used for analysis of phosphoproteomes of other proteome samples.
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