Proteomics Research On Secreted Proteins And Glycoproteins Of Human Hepatocellular Carcinoma Cells

The main contributions of this dissertation were: to solve the difficulties on secretome research, a series of studies have been conducted. For the enrichment of secretome in large volume of growth media rich in salt, a simple, fast, effective and universal approach was developed and applied well on secretome research of HCC cells. And highly-efficient enrichment for the low molecular weight proteins (LMWPs) in secretome has been achieved via a specially designed experiment. Notably, the smallest MW touched 4 KDa. A secretomic profile of HCC cells was built up, and this database would exhibit valuable information in the research of HCC metastasis. A large-scale detection of glycosylation sites of secreted proteins was successfully performed. 172 new N-glycosites were determined experimentally, including the new N-glycosites of CD44 and laminin etc, which have been reported to be implicated invasion and metastasis of HCC. Importantly, the utility of N-glycoproteomic strategy has been proved to be a great way to profile the genuinely secreted proteins. The fact that 96.4% of the identified glycoproteins could be secretory proteins positively confirmed this good point. To get the information including molecular function, signal pathways and protein-protein interactions, bioinformatic analyses on the secretome identified were conducted deeply. To exam the reliability of glycosites identified according to the mass increased of 1 Da (¹⁶O labeled) or 3 Da (¹⁸O labeled), the excellent consistence of glycosites from two labeled was founded. Thus, a frequently asked question was answered by experimental data for the first time.Given the importance of secreted proteins as a source for early detection and diagnosis of disease, secreted proteins have been arousing considerable attentions, and the field of secreted proteome (secretome) has achieved substantial advances during recent years. In addition to the comparative proteomic analyses focusing on the characterization of differentially expressed proteins in two different, well controlled states, e.g. normal and disease, the global profiling of secretome received considerable attraction. However, the analysis of secreted proteins represents some challenges in four aspects as following. 1) The contamination of the authentic secreted proteins is unavoidable, resulting in severe interferences for the identification of secreted proteins; 2) those proteins with low molecular weight or with low abundance are difficult to be discovered; 3) little attention has been paid to the post-translational modifications (PTMs) of the secretory proteins; 4) the functional investigation of those detected secreted proteins has been also paid little attention. To find solutions for the questions mentioned above, a series of researches have been performed and some satisfactory results have been achieved.Due to vital roles of glycosylation of proteins in lots of important physiological and pathological processes, interest in investigating glycosylation status of glycoprotein has greatly increased in recent years. Because the great progresses of techniques including enrichment methods for glycoproteins/glycopeptides and mass techniques for biomolecules have been made, the study of glycoproteome is progressing at tremendous speeds and has achieved substantial advances during recent years, special in the large-scale detection of glycosylation sites. N-glycoproteins and classical secreted proteins have the same dependence of the endoplasmic reticulum (ER) - Golgi network, and the classical secreted proteins are almost N-glycoproteins. Thus, a large-scale glycoproteomic identification and N-glycosylation site elucidation has been performed for secreted proteins of HCC cells. Also, the glycoform has been analyzed elementally.How tumors spread and kill their host organism remains an enigma, but not for lack of attention. A renewed focus on the problem of metastasis is now apparent, and for good reason—metastasis remains the cause of 90% of deaths from solid tumors. Hepatocellular Carcinoma (HCC) is a common malignancy worldwide and is a second leading cause of death in our country. To understand better the mechanism of HCC metastasis, the human HCC metastasis nude mice model and HCC cell lines with different metastasis potential were built up by the Liver Cancer Institute of Zhongshan Hospital, Fudan University. This work is made on the two HCC cell lines (HCCLM3 and MHCC97L) with different metastasis potential. The investigation has focused on the application of proteomic and glycoproteomic methods in the secretomic research, and a lot of valuable proteome information has been obtained.This dissertation consists of 7 parts and the contents are summarized as follows:In the first chapter, the seven aspects of secretomic research were summarized, also, the research progresses and the bottle neck. In addition, the advances in the research of glycosylation were summarized, specially, in the separation and enrichment of Glycoproteins/Glycopeptides. Furthermore, a review of liver proteomic study was presented. Importantly, the aims and significance of this dissertation were presented.In the second chapter, primary research of secretome of HCC cells has been made. In the first section, three most often used concentration methods (ultrafiltration, dialysis and precipitation) were used together and compared. The merits and shortcomings of each method were discussed based on the obtained results. Four aims of this section have been achieved: the optimized method for cell culture in conditioned medium was developed; three concentration methods (ultrafiltration, dialysis and precipitation) were compared; the secretome of HCC cells with metastasis potential was studied primarily; the bottle neck of secretomic research was founded our. And all these works were basic for further research.In the third chapter, a simple, fast, efficient and universal enrichment process for secretome was developed. Nanozeolite LTL was used to capture secreted proteins for the first time. Highly-efficient enrichment for the low molecular weight proteins (LMWPs) in secretome has been achieved, and attracted most of attentions in a specially designed experiment. Followed by 1D SDS-PAGE for protein fractionation and then by LC-ESI-MS/MS for protein identification. Totally 1474 unique proteins were confidently identified and 97 proteins of 1474 proteins were those notably with MW less than 15 KDa, which were seldom captured previously by traditional methods. Notably, the smallest MW touches 4 KDa. Many proteins identified above were found to be involved in the processes associated with the cancer development and metastasis. For instance, alpha-fetoprotein (AFP), a widely known biomarker of HCC, was detected. Osteopontin, a secreted phosphoprotein, is a significant factor in HCC metastasis, and its over-expression correlates with metastatic potential of primary hepatocellular carcinoma, and with invasiveness of liver tumor-derived cell lines in vitro. Elevated levels of matrix metalloproteinase (MMPs) have been shown in many tumors with strong association with the invasive and metastatic potentials. These observations demonstrate the contention that secretomic approach is a great way to the discovery of potential biomarkers. And the strategy that started with a capture of secreted proteins by nanozeolite LTL, followed by a separation of 1D SDS-PAGE and by an identification of LC/ESI-MS/MS has been proven to be perfect. Notably, for special enrichment of LMWPs, compared to conventional methods, this strategy was special and efficient.In the fourth chapter, glycosylation status of HCC cells was investigated. In the first section, a large-scale detection of glycosylation sites of secreted proteins of HCC cells was successfully performed. For the enrichment of glycopeptides, capture methods with hydrophihc affinity (HA) and hydrazide chemistry (HC) were used complementarity. Using both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously, including the new N-glycosites of CD44 and laminin etc, which have been reported to be implicated invasion and metastasis of HCC. Importantly, the utility of N-glycoproteomic strategy is a great way to profile the genuinely secreted proteins. The fact that 96.4% of the identified glycoproteins could be secretory proteins positively confirmed this good point. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% VS 51.3%), however, based on the number of glycosites identified, HA outweighs HC (265 VS 159). This result based on the compared experiment could be useful for the selection of methods. In the second section, glycoforms of secretome of HCC cells were profiled primarily by multi-lectin blot. The difference of glycosylation status of two HCC cell lines (HCCLM3 and MHCC97L) with different metastasis potential were compared primarily, and based on these results more researches would be done in the near future.In the fifth chapter, to get the information including molecular function, signal pathways and protein-protein interactions, bioinformatic analyses on the secretome identified were conducted deeply. Ingenuity Pathway Analysis system was used to excavate the secretome data from chapter two, chapter three and chapter four. The sub-network, protein-protein interactions, cell-cell interactions and crucial pathways were investigated. Also, some key node molecules were discussed. All the explorations are basic and helpful for HCC research in future.In the sixth chapter, to exam the reliability of glycosites identified according to the mass increased of 1 Da (¹⁶O labeled) or 3 Da (¹⁸O labeled), deglycosylation experiments were carried out parallelly H₂¹⁶O in or H₂¹⁸O. As a result, the excellent consistence of glycosites from two labeled was founded. Thus, the results from ¹⁶O labeled were proven to be credible, and a frequently asked question was answered by experimental data for the first time.In the last chapter, a summary was made and a prospect was presented.