Human Liver Cona And Dsa Bound Glycoproteins Group Study

Part OneCon A Affinity Glycoprotemics of Normal Human Liver TissueObject: For the rapid progress in proteome research, study on post translation modification of proteins including protein glycosylation has been attracted more attention. In this part, Concanavalin A (Con A), a lectin from Canavalia ensiformis, was employed to enrich the special N-glycoproteins with complex and high-mannose-type oligosaccharide chains of normal human liver tissue by affinity chromatography. Thereafter the Con A affinity glycoproteins were separated and profiled by 2-DE and the visible protein spots were further identified by mass spectrum (MS) analysis. A stable and standard glycomic approach was constructed for analyzing normal liver tissue proteins and the establishment of Con A affinity glycoprotein profile and database could give us a help for post work.Methods: We purifed and concentrated the glycoproteins with high-mannose core and the two antennary type by the ConA affinity chromatography. Using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based multiplexed proteomics (MP) technology, ConA affinity protein expression profile of normal human liver tissue were generated and analyzed. The visible glycoprotein spots on the gel were identified by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS). The glycosylated-sites were predicted with NetNGlyc1．0 and NetOGlyc3．1 software. Meanwhile according to the geneontology methods, they were classified by molecular function biological process and cellular component.Results: Proteins extracted from normal liver tissues were separated and condensed into two fractions,ⅠandⅡFractionⅠmainly consisted of non Con A affinity glyproteins, and fractionⅡenriched in Con A affinity glyproteins. Two fractions were subjected to 10% SDS-PAGE. In fractionⅡ, It was found that the low abundant protein could be highly enriched by lectin affinity chromatography for example Con A affinity proteins located 60kDa position was a band with high intensity but it was not found in unbound fraction. Con A affinity glycoprotein expression profile of normal human liver tissue was obtained by 2DE separation and following SYPRO-Ruby staining (Fig3) . 301±15 of glycoprotein spots were visible and analyzed by PDQuest software. Con A affinity glycoprotein centralized between the acidic and basic region. High abundant proteins distributed mainly in the area of pH 3-5, MW 25-47KD. Glycoproteins with low molecular weight were also visible. 139 of 301 glycoprotein spots were cut out and identified via MALDI-TOF-MS/MS. Database searching was performed using routine procedure which IPI. Finally 85 glycoproteins from normal human liver tissue were successfully identified. We applied post-translational modification prediction software in ExPASy Proteomics Server (http://ca.expasy.org/tools/#ptm), namely NetNGlyc and NetOGlyc to predict N-glycosylation sites and O-GalNAc (mucin type) glycosylation sites. 5 proteins only had potential O-linked glycosylation sites and all of others contained potential N-linked glycosylation sites. These identified proteins were then classified bioinformatically and we found that they mainly distributed in organelle, participated in the metabolism, and played crucial roles in binding and catalytic reactions.Conclusion: (1) Lectin affinity chromatography coupled with 2-DE and MALDI-TOF-MS/MS was the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics. It can be used for the research of diseased glycoproteomics with large sample size and play an important role in finding abnormal glycoproteins which were related with disease development. (2) We constructed the Con A affinity glycoprotein profile and database. 85 glycoproteins from normal human liver tissue were successfully identified. According to the sub-cellular localization, Con A affinity glycoproteins distributed in cytoplasm, nuclear and cell membrane at different level. Participated in the metabolism, and played crucial roles in binding and catalytic reactions.Part Two DSA Affinity Glycoproteomics of Human Liver TissueObject: In this part, Datura Stramonium Agglutinin (DSA), a lectin from Datura stramoniumL. (Jimson weed), which can strongly bound to CC triantennary GlcNACManGlcNAC and weakly bound to CC triantennary GlcNACManGlcNAC, was employed to enrich the special N-glycoproteins of normal human liver tissue and liver cancer tissue by affinity chromatography. Thereafter the DSA affinity glycoproteins were separated by 2-DE and two-dimensional Liquid Chromatography (2D-LC), then identified by MALDI-MS-MS and ESI-MS. The DSA affinity glycoproteins database of normal human liver tissue and liver cancer tissue were constructed, which will provide us some basic information of total glycoproteins in normal liver tissues and contribute to discovering biological functions of triantennary sugar chain in vivo.Methods: DSA glycoprotein's expression and location were proved by immunofluorescence (IF). The glycoprotein with triantennary type were purified and concentrated by the DSA affinity chromatography. One fraction was using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by multiplexed proteomics(MP) technology to get the glycoprotein expression profiles, The detection of spots and comparison of glycosylation extent were conducted with 2-DE analyzing software Image master 6．0．Glycoprotein spots were characterized by matrix assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF-MS). Another fraction was separated and identified by 2D-LC coupled ESI-MS. The two separation methods were compared. The potential proteins' glycosylation sites were predicted with NetNGlyc 1．0 and NetOGlyc 3．1 software; meanwhile they were classified according to the geneontological methods.Results: We found DSA affinity glycoprotein located in membrane, cytosol and nuclear in Chang's liver cell. The DSA elution glycoproteins were successfully purified from the total protein of normal human liver tissue and liver cancer tissue by 10% chitin. The chromatography yield of DSA affinity glycoproteins was 4% with the comparison of loaded total liver protein. The PAGE profile of DSA-binding protein from normal liver tissue was significantly different with that of liver cancer tissue. DSA affinity glycoprotein expression profile of normal human liver tissue was obtained by 2-DE separation and following Pro-Q 488 staining. 60±3 of glycoprotein spots were visible and analyzed by Image master software. DSA affinity glycoprotein centralized between the acidic regions. High abundant proteins distributed mainly in the area of pH 4-7, MW 35-85KD. 43 glycoproteins from normal human liver tissue were successfully identified via MALDI-TOF-MS/MS. 93 glycoproteins from normal human liver tissue were successfully identified via 2D-LC coupled ESI-MS. 25 of them were also detected by 2DE-MALDI-MS. The glycoproteins which were detected by 2D-LC were centralized between the acidic regions. But their molecular weight were more wider than those who were detected by 2DE-MALDI-MS. 98 glycoproteins from liver cancer tissue were successfully identified via 2D-LC coupled ESI-MS. 73 of them were matched with that of normal liver tissue under the same conditions. 45 of them were unmatched with that of normal liver tissue. Most of the identified glycoprotein had potential N-linked glycosylation sites. DSA affinity glycoproteins distributed in cytoplasm, nuclear and cell membrane at different level, participated in the metabolism, cell adhension and cell motility. More DSA affinity glycoproteins were located in endocytoplasmic reticulum(ER) and play an important role in protein folding.Conclusion: (1) Lectin affinity chromatography coupled with 2-DE, MP technology, MALDI-MS or Lectin affinity chromatography coupled with 2D-LC-ESI-MS were the two high throughput, high efficiency glycoproteomics platform. 2D-LC can be used in small sample size. (2) DSA affinity glycoprotein profile and database were constructed in normal and cancer tissue. 118 glycoproteins from normal human liver tissue were successfully identified by two methods. 98 glycoproteins from liver cancer tissue were successfully identified by 2D-LC-ESI-MS. According to biological analysis, DSA affinity glycoproteins maybe participated in the cell adhension, protein folding and transportation.Part ThreeDSA affinity Glycoproteomics Correlated with HCCObject: In this part we applied two dimensional differential in gel electrophoresis (2D-DIGE ) to compare the profile of DSA affinity glycoprotein in normal liver tissue and liver cancer tissue in order to find out and identify the HCC biomarker. We choose one protein to verify the expression and predict the sugar chain structure.Methods: Normal and cancer DSA affinity glycoprotein were labeled with Cy3 or Cy5 randomly while internal standard pooled sample was labeled with Cy2 dye. Samples were finally combined according to the experimental design. Fluorescence images of the gels were acquired on a FuJI 5100 scanner. Cy2, Cy3 and Cy5 images were scanned at 473nm, 532nm and 635nm. Image analysis and statistical quantification of relative protein abundances was performed using DeCyder 2D 6．5 software. Interesting differential spots were manually excised from each of two preparative gels and identified via MALDI-MS. The identified differential glycoprotein including those who were detected by 2D-LC-ESI-MS, were biologically analyzed via their function and pathways. Vimentin, one kind of the differential glycoprotein, was selected for further analysis by western blot, immunofluorescence, immune precipitation and lectin blot. Results: The gels were respectively imaged by FUJI 5100 using different emission filters and spot features were analyzed by DeCyder 2D 6．5 software, in sum, 308±15(n=3) , 353±26(n=3) protein spots were respectively detected in normal and cancer, among them 266 protein spots were well matched. 41 protein spots were unmatched and concluded to be the impure substance or probable error. 30 protein spots showed 1．4 or more than 1．4 fold differences and then cut out. 26 differential proteisns were identified, combining the differential glycoproteins which were identified in Part 2．Biological analysis showed that most differential glycoprotein were located in ER or Golgi and dedicated in protein folding, post translational modification and ER stress. Vimentin was confirmed to be highly expressed in cancer by western blot. It had a higher affinity for DSA and Con A in cancer and had no difference for bound for LCA in normal and cancer tissue.Conclusion: (1) Lectin affinity chromatography coupled with 2D-DIGE and MALDI-MS was the novel high throughput and high efficiency comparative glycoproteomics' technological platform. (2) 16 DSA affinity glycoprotein's expression was positively in correlated with HCC development. (3) Vimentin was proved to have high mannose-core, triantennary type and core-fucosylated type sugar chain structure.The potential application of this work1．The establishment of DSA and Con A glycoprotein database from normal liver tissues could provide us some basic information about biological functions of sugar chain in vivo.2．Some of identified aberrantly DSA affinity proteins might become biomarkers for diagnosing and predicting HCC prognosis.Novelty of the project1．We established two high throughput glycomic methods on the basis of 2D-DIGE and 2D-LC, which would lay sound foundation for the studies on glycoproteomics related with the occurrence and development of some diseases.