Study On Phosphorylated Proteomics Of Different Growth Factors Stimulating Phosphatidylinositol - 3 Kinase Signaling Pathway

Proteins are the fundamental base of biological activities in livings organisms. Proteins perform a vast array of functions, including transporting molecules, catalysis, regulation, immunity, and control in biological process. Proteomics, in a way of high through-put and sensitivity, was developed to globally and deeply research on proteins which were expressed from genomics. As a result, we could get more systematic and in-depth knowledge about livings.Phosphorylation, as one of the most important and global post-translational modification in proteomics, takes a crucial role in life process of majority of cells. The phosphorylation of proteins is closely related to metabolism, material transporting, signal transduction, proliferation, differentiation, and apoptosis of cell and tumor physiopathology. Phosphatidylinositol3-kinase signaling pathway participated in many physiological and pathological processes, considered to be a tumor-specific potential drug target. With its further study, researchers found that many problems remain unresolved. For example, through clinical trials, potential issues associated with toxicity and resistance can be expected. PI3K mutants that are resistant to a kinase inhibitor may arise during treatment, or other parallel and/or interconnected pathways, may also render cancer cells resistant to PI3K inhibition. It is therefore important to identify new therapeutic targets for the development of drugs that may be used either in place of PI3K inhibitors or to enhance the efficacy of PI3K inhibitors at subtoxic doses.This thesis is a cross-linking research related on chemical biology, cell biology and analytical chemistry. With the combination of differential proteomics and phosphoproteomics based on mass spectrometry, it was quantified successfully for the pathway of PI3K stimulated by different growing factors. Several differential expressed proteins were identified. In conjunction with research on lipidomics, the regulating rule of PI3K was discovered and analyzed.Main contents in the thesis are as follows:The first chapter is the introduction part. The advance in proteomics and phosphoproteomics research and the background of PI3K pathway was reviewed.The second chapter is about optimization for phosphopeptides enrichment strategy. Standard proteins and mouse liver were used as model samples for phosphopeptide enrichment.The third chapter is the relative quantitative analysis of PI3K pathway that stimulated by different growing factors. A variety of strategies including SCX, label-free quantitation and IMAC were applied for phosphoproteome analysis. As a result, a total of1598phosphopeptides have been identified and quantified.250phosphopeptides were identified in control, whereas1409phosphopeptides were found in EGF sample. Of the1409peptides,1340phosphopeptides corresponded to842phosphoproteins were up-regulated. On the other hand,729phosphopeptides were identified in PDGF sample, which566of them corresponded to386phosphoproteins were detected as up-regulated. Moreover, of these729phosphopeptides,139are unique phosphopeptides that were only identified in PDGF sample, which corresponded to122phosphoproteins. For EGF sample,784phosphopeptides corresponded to756phosphoproteins were identified as unique peptides. Both the unique phosphopeptides from EGF sample and PDGF sample were deeply analyzed, indivually, by sequence analysis and IPA analysis. The common phophoproteins between EGF sample and PDGF sample aslo analysed by IPA analysis.The main contribution and innovation of this thesis are:1) thorough understanding of the limitation of existing phospho-enrichment and data analysis in our laboratory, offering an improved process and fully utilization of enrichment of phosphopeptides.2) bio-informatics analysis for data from phosphoproteome. With label-free quantative analysis and IPA analysis. Differential expressed phosphopeptides have been quantified, and a number of potential pathways have been revealed. It is the first time to connect systematic biology to lipidomics.