Development Of The Qualitative And Quantitative Phosphoproteomics Methods And Their Application In Liver Cell Phosphoproteome Analysis

Protein phosphorylation plays an essential role in regulation of cellular actions and recently, phosphoproteomics is becoming a hotspot. The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a large scale is essential for biological and clinical research. Protein phosphorylation on a proteome wide scale still poses substantial challenges with respect to the frequently low abundance and substoichiometric phosphorylation-site occupancy of phosphoproteins. Liver, as the body's internal chemical power plant, performs many complex and essential functions, which are primarily regulated via reversible phosphorylation. The phosphoproteome analysis of the liver cell allows us to further comprehend and recognize the liver's complex function and the essential regulation of phosphoprotein in it.This study analyzed the phosphoproteome of the liver cell by qualitative and quantitative technology.In the first part, standard proteins mixture from yeast were respectively used to optimize the SCX-LC-MS/MS strategy, and then the SCX and a complementary mass spectrometric strategy were used to characterize the phosphoproteome of the human Chang liver cell and probe the role of protein phosphorylation in these cells. In total 559 phosphorylation sites and 409 phosphorylated peptides were identified from 370 phosphoproteins in human Chang liver cell. Compared to the public databases of UniProtKB/Swiss-Prot, approximately 255 phosphoproteins and 271 exact phosphorylation sites are novel. Most of them play an essential function closely connected with the physiological and pathological mechanism in human liver. The resulting dataset is the first report on the phosphoproteome of a human normal hepatic cell, which will provide further insight into comparison with liver cancer cells. In the second part, ¹⁸O -labeling strategy were established and optimized. And then we established the integrated enrichment strategy of phosphoproteins by combining two enrichment methods (IPG-IEF and TiO₂) and LTQ-FT MS with the higher accuracy and speed. After comparing with other enrichment strategy, this combined enrichment strategy was considered to be effective in separating phosphopeptides and well compatible with ¹⁸O-labeling, the study above has not been reported before. A computational tool named MSOQ was developed to automate the calculation of the relative intensity of each ¹⁶O- and ¹⁸O labeled. And it could also help to obtain an accurate measurement. HBx is a recognized risk factor in the development of hepatocellular carcinoma (HCC), which had been paid close attention. The established ¹⁸O-IPG-IEF-TiO₂-LTQ-FT strategy above was developed to the quantitative phosphoproteomic analysis of the HepG2/HepG2-HBx. Totally, 1358 phosphosites, 958 phosphopeptides and 895 phosphoproteins with 1% false positive ratio were identified, and compared with UniProtKB/Swiss-Prot, approximately 85% phosphoproteins and more than 90% phosphorylation sites are novel. In view of the fact that N-terminal phosphorylated peptides are the greatest proportion of identified phosphopeptides in chang cell and HepG2 cell, we presumed that phosphorylation is probably more likely to occur in the flexible and exposed N- region of a protein. With respect to quantitative analysis, 157 phosphosites, 182 phosphoproteins and 1362 nonphosphoproteins were found to be modulated by HBx. We also found that there were no changes in proteins abundance but great difference occurred in phosphorylation modifications. These results would allow further insight into the mechanism of HCC caused by the integrated HBx and may help to search some biomarkers for HCC.