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Phosphorylated protein analysis method and its phosphorylation in human fetal liver proteome study

Author: WangJingLan
Tutor: QianXiaoHong
School: PLA Military Academy of Medical Sciences
Course: Drug analysis
Keywords: Phosphoproteome Biological Mass Spectrometry Immobilized metal affinity chromatography Human fetal liver Membrane protein
CLC: R341
Type: PhD thesis
Year: 2004
Downloads: 423
Quote: 1
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Abstract


Phosphorylation is one of the most important vivo protein post-translational modification, protein phosphorylation and dephosphorylation process is the most common means of regulation of eukaryotic cellular activities. On the determination of the detection of the phosphorylated proteins and phosphorylation sites recognize the important reference information for protein phosphorylation in the regulation mechanism in the life activity, proteomics strategy Graduate object of the phosphorylation modification of functional proteomics research content . The phosphorylated proteins in vivo content is very low, its detection and site analysis has been a difficult task, and large-scale analysis and identification of sites phosphorylated proteins phosphorylation of Proteome Research technical challenges. In this paper, the work includes phosphorylation proteome research based on the establishment of affinity extraction and biological mass spectrometry phosphorylated protein analysis methods, as well as the large-scale protein phosphorylation analysis techniques in human fetal liver. The first chapter sample protein phosphorylation analysis technology system: standard phosphorylated proteins immobilized metal affinity chromatography based phosphopeptide affinity enrichment technology help from the complex peptide mixture of phosphoric acid peptide candidates ; MALDI-TOF spectrum phosphopeptide metastable ion peak auxiliary judgment serine / threonine phosphorylation type; phosphopeptide candidate phosphatase hydrolysis experiments combined with mass spectrometry can prove the presence of phosphoric acid peptide; Finally, tandem mass spectrometry analysis combined with database searches identification used to determine the phosphorylation sites. The second chapter to build a full automation of solid-phase metal affinity chromatography - reversed-phase liquid chromatography - tandem mass spectrometry phosphorylated protein analysis platform, process control, convenient, easy to change the experimental conditions, depending on the sample and optimization of analysis parameters. The standard phosphopeptide investigated the performance of the system, to optimize the analysis parameters and applied to the identification of the recombinant human TRF1 in vitro phosphorylation sites identified two phosphorylation sites for protein kinase phosphorylation provides useful The information given. Chapter III of the paper using the phosphorylated protein analysis methods established large-scale analysis of the human fetal liver cell membrane to extract proteins phosphorylated proteins were identified 28 phosphopeptides, 36 phosphorylation sites were attributable to the 21 phosphate of protein, of which 27 phosphorylation sites are not reported in the literature before. Carried out for the identification of protein phosphorylation sites prediction, and were compared with the phosphorylation sites identified two phosphorylation sites prediction software. Finally, phosphorylation of proteins identified subcellular localization and functional classification analysis.

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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