Glycoproteome Investigation Of Human Liver Cells And Tissue

Part OneObject:To construct the glycoprotein profiles and glycoprotein databases of normal human liver cell line (Chang's liver) and human liver cancer cell lines with differently metastatic potential(Hep3B and MHCC97H), and to compare the differences of glycosylation status between three cell lines. Methods: Total proteins were extracted respectively from three cell lines, then subjected to two-dimensional electrophoresis (2-DE). Finally 2-DE gels were stained according to the methods of multiplexed proteomics(MP) technology to get the glycoprotein expression profiles and total protein expression profiles of three cell lines. The detection of spots and comparison of glycosylation extent were conducted with 2-DE analyzing software Dymension. Glycoprotein spots were characterized by matrix assisted laser desorption/ ionization-time of flight mass spectrometry(MALDI-TOF-MS). Results: The glycoprotein profiles of three cell lines were obtained through 2-DE combined with MP technology. Dymension software detected glycoprotein spots 74±2(n=3) in the 2-DE gels of sample from the normal human liver cell line Chang's liver, 78±3 (n=3) in the 2-DE gels of sample from the non-metastatic human liver cell line Hep3B and 72±5(n=3) in the 2-DE gels of sample from the highly metastatic human liver cell line MHCC97H. The comparison betwween two groups were proceeded with the best run gels, Hep3B group as reference gels. Comparing with Hep3B, Chang's liver group had average 31±3 and MHCC97H group had average 36±4 matched glycoproteins spots. Mass spectrometry had successfully determined 63, 59 and 52 glycoproteins from Chang's liver, Hep3B and MHCC97H respectively. In addition, Dymension software analysing shows that three cell lines have not only significantly diverse glycoprotein profiles, but also significant differences of glycosylation extent of proteins. Comparing Hep3B with Chang's liver, 25 glycoproteins have the alteration of glycosylation extent, on the other hand, comparing MHCC97H with Hep3B, 29 glycoproteins have the alteration of glycosylation extent. Conclusion: MP technology is high-throughput sensitive method detecting glycoproteins in gels, and has excellent compatibility with mass spectrometry. 2-DE, MP technology combined with MS formed a novel research platform that constructs glycoprotein profilesor glycoprotein databases. This study also demonstrated that not only differential glycoproteins, but also the alteration of glycosylation extent of glycoproteins are correlative with the carcinogenesis, development and metastasis of liver cancer, andmay play the critical roles.Part TwoObject: To extensively investigate the glycoproteins of normal human liver tissue, including the glycoprotein expression profile and database. Methods: Total proteins were extracted from the normal human liver tissue, then subjected to two-dimensional electrophoresis (2-DE). Finally 2-DE gels were stained according to the methods of multiplexed proteomics (MP) technology to get the glycoprotein profiles and total protein profiles. Detecting and matching spots were conducted with 2-DE analyzing software PDQuest. Glycoprotein spots were excised and then characterized by matrix assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF-MS). Results: PDQuest software detected glycoprotein spots 1011 as well as total protein spots 1923 in the 2-DE gels of sample from the normal human liver tissue. Furthermore, 116 glycoproteins were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS, and annotated to our databases. Conclusion: This study demonstrates the feasibility of the novel technological platform to contruct glycoprotein databases. These results pour new information into burgeoning glycoproteomic fields andlay a foundation for future physiological and pathological studies of human liver. Part ThreeObject: To exhume more potential markers for diagnosing HCC and predicting HCC metastasis by analyzing our constructed databases using bioinformtic tools and resources.Methods: utilizing web-accessible bioinformatic database resources bioinformatic software to analyze glycosylation sites and founctions of glycoproteins,draw glycoprotein interaction network and predict signal peptides of glycoproteins. Results: (1)Each protein in our constructed databases have potential glycosylation sites and approximately 90% of them were predicted to be glycoproteins.(2)Comparing normal cell line with non-metastasic and high-metastasic liver cancer cell lines, differentially expressedglycoproteins include stress proteins, Redox-regulating proteins , nucleic acid synthesis-associating proteins, cytoskeletal proteins and proteinase.(3)The protein interaction networks of three cell lines show that the differentially expressed glycoproteins interact with groups of protein relating to proliferation and polarization of cells.(4) 11 species of differentially glycoproteins were predicted to be secretive proteins.Conclusion: Some glycoproteins combine with groups of proteins relating to proliferation and polarization of cells to form a giant network of interaction, which result in evil proliferation of cells. The notion "protein groups" may be introducted to diagnosis and prediction of HCC metastasis. Toward the differentially expressed glycoproteins containing signal peptides, serological validation will provide more potential biomarkers for diagnosing HCC and predicting HCC metastasis. In addition, alteration of their glycosylation also may be novel glycome markers for diagnosing HCC and predicting HCC metastasis. The potential application of this work:1 Be potential biomarkers for diagnosing HCC and predicting HCC metastasis and recurrence and for progonosis of HCC.2 To provide glycome markers for diagnosing HCC and predicting HCC metastasis and recurrence with glycosylation abnormity of glycoproteins.Novelty of this project:1 Utilizing proteomic platform combined with glycoproteomic technology, namely 2-DE,MP and mass spectrometry , primarily construct high-efficiency, high-sensitivity and high-throughput technologic platforms for researching glycosylation profiles of proteins.2 Using bioinformatic tools and databank resource to analyze glycoproteomic data, and discovering significant glycoproteins in incurrence and metastasis, moreover potential glycome markers for diagnosing HCC and predicting HCC metastasis...