Phosphoproteome Analysis Method In The Human Fetal Liver Phosphoproteome Applications

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 such as cell cycle, differentiation and development, metabolism, nerve activity, muscle constriction, and transcriptional regulation. So detecting phosphoproteins and defining their phosphorylation sites are very important to understand the mechanism by which phosphorylated protein affects a biological pathway. Analysis of the entire complements of phosphorylated proteins in cells, or the 'phosphoproteome', has become a key part of functional proteomics recently. However, because the stoichiometry of phosphorylation 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. We report here the setting up of comprehensive phosphoprotein analysis methods based on the combination of affinity chromatography and mass spectrometry. And we also report the phosphoproteomics analysis of membrane proteins from human fetal liver.In chapter one, systemic analysis methods for phosphoproteins are established. Immobilized metal affinity chromatography (IMAC) is used to selectively enrich phosphopeptides from protein digest mixtures. Metastable ion peaks of phosphopeptides in MALDI-TOF-MS spectrum are identified for the judgement of S/T phosphorylation type. Treatment of phosphopeptides with alkaline phosphatase and analysis of the peptides before and after treated with mass spectrometry is used to confirm the phosphorylation of the peptides. Finally, the phosphorylation sites are determined by tandem mass spectrometry analysis and database searching.A robust and automatic IMAC-RPHPLC-MSMS system is developed in chapter two. The system is optimized by analyzing standard phosphopeptide, and then is applied to the identification of phosphorylation sites of rhTRFl treated with kinase in vitro, and two phosphorylation sites are defined.Phosphoproteomics analysis of membrane proteins from human fetal liver are reported in last chapter. A total of 21 phosphoproteins, 28 phosphopeptide sequences are determined, 36 phosphorylation sites are defined, and 27 of those sites are newly identified. Two softwares are used to predict the possible phosphorylation sites of identified proteins and the predicted results are compared with those identified. All the phosphoproteins identified from the membrane protein of human fetal liver are categorized according to their functions and the localization of these proteins in cell is analyzed.