| Protein glycosylation is one of the most important and common post-translational modifications. It not only plays a key role in protein stability, solubility, folding, and activity, but also participates in cellular processes such as protein trafficking, sorting and molecular recognition. Protein glycosylation also plays a key role in the infected process of influenza viruses. It affect the host range and virulance of the virus. In addition, the aberrant glycosylation is a fundamental characteristic of cancer and other disease states, and many clinical biomarkers and therapeutic targets are glycoproteins. Thus, the development of new technologies for protein glycosylation research as well as the comparison analysis of protein glycosylation among samples at different physiological and pathological states at glycomics level are becoming more and more important in glycoproteomics as they can help us to obtain a better understanding of the crucial functions of protein glycosylation, to find some new potential diagnostic markers and therapeutic targets and to investigate the molecular mechanism with regard to various kinds of physiological and pathological states.Here, the first part of the research was to develop a noval method by modifying magnetic particles with hydrazine chemistry for simple and rapid isolation of N-glycopeptides from biological samples and further to develop a new system with the combination of mass spectrometry for identification of N-glycosylation sites (N-glycosites). After the method selection for preparation of hydrazine-functionalized magnetic particles and optimization of reactive conditions (such as reactive time and temperature) for isolation of N-glycopeptides, the N-glycopeptides capability and specificity of hydrazine-functionalized magnetic particles were higher than hydrazide resin. The feasibility of hydrazine-functionalized magnetic particle in isolating N-glycoproteins/glycopeptides and finally identifing were confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites.The second part of the research was to isolate formerly N-glycopeptides from hepatocellular carcinoma (HCC) patients sera as well as healthy volunteer sera by employing N-glycoproteins isolation,N-glycopeptides isolation and sialic acidic N-glycopeptides isolation methods based on hydrazide chemistry and further to identify N-glycoproteins and N-glycosites with the assistance of mass spectrometry. Meanwhile, the N-glycoproteins in HCC and healthy human sera were quantitied using emPAI label-free method by merging the MS/MS data of nonglycopeptides and glycopeptides from glycoproteins isolation method for database search (modified N-glycoprotein isolation method). A total of 167 unique N-glycopeptides which contained 183 N-glycosites and 103 glycoproteins were identified from HCC and healthy human sera, in which 44.7% of N-glycoproteins and 48.0% of N-glycopeptides were sialic acidic,15% of N-glycoproteins and 17.7% of N-glycopeptides were identified at the first time. The serum N-glycoproteins identified in the experiment were mainly at excellular regions, cell, organelle, membrane-enclosed lumen and macromolecular complex; mainly take part in the biological process of response to stimulus, biological regulation, cellular process,multicellular organismal process, immune system process, metabolic process and signaling, and play the molecular functions of binding, enzyme regulator activity and catalytic activity. Of the identified N-glycoproteins, seventy six were identified in both type of sera, twelve were only identified in HCC patients sera and fifteen were only identified in sealthy volunteer sera. In addition,9 N-glycoproteins were markedly higher and 7 were lower in HCC petients sera than in healthy volunteer sera. These differential N-glycoproteins were mainly take part in the defense reponse, cellular process, regulation of cellular process, regulation of biological process, development process etc. Besides, the results also showed that the modified N-glycoprotein isolation method possesed the advantages of making the identification of glycoproteins more accuracy, emPAI label free method can be employed for quantification of glycoproteins, identifying many N-glycoproteins which can not be identified in N-glycopeptide isolation method and complementing N-glycopeptide isolation method well. The data here can provide scientific evidence and reference for the research of HCC molecular machanism and finding research of potential diagnostic markers and therapeutic targets of HCC.The third part of the research was to isolate formerly N-glycopeptides from healthy human whole saliva of different gendar and ages (chirdren, adult and the older,6 groups) by employing hydrazide chemistry and hydrophilic method, and further to identified, comparised and compr analyzed human whole saliva N-glycoproteins and N-glycopeptides. A total of 156 unique A'-glycopeptides which contained 164 N-glycosites, represented 103 glycoproteins were identified from 6 groups of whole saliva, of which 18% of N-glycoproteins and 24% of N-glycopeptides were identified for the first time,and 17 known N-glycoproteins were identified in human saliva for the first time.41.2% of saliva N-glycoproteins and 35.2% of N-glycopeptides also exist in human serum. The molecular weight of 78% identified saliva N-glycoproteins were at 10-100KD and 80.2% were pI<7, indicating that the saliva N-glycoproteins are mainly medium or low molecular weight and acidic proteins. The saliva N-glycoproteins identified in the experiment were mainly at excellular regions, cell and organelle; mainly take part in the biological process of cellular process, response to stimulus, biological regulation, metabolic process and immune respect; and function as binding, catalytic activity and enzyme regulator activity. The types of N-glycoproteins in human whole saliva increasing as the people grew old, and the rate was bigger in male than in female. The difference of the type of saliva N-glycoproteins was the biggest between adult male and female and was the smallest between the older male and female. Many age and gendar associated glycoproteins were found in human saliva. The new N-glycoproteins added in saliva as people grew old mainly take part in the molecular functions of the negative regulation of immune respect, multicellular organismal process, regulation of enzyme inhibitor activity, positive regulation vascular endothelial growth factor production and biological process as well as regulation of cell death and programmed cell death. The increase of immune associated N-glycoproteins was happened in the grow process of age of both male and female, and these N-glycoproteins mainly take part in the complement cascades.41.2% of salivary N-glycoproteins also exist in human serum. Besides, the results also showed that both capability and specificity of N-glycoprotein isolation by hydrazide chemistry were more prominent than by hydrophilic method, but there was still complementarity between two methods. The data here could provide scientific evidence and reference for the proteomics research of human saliva associated with gendar and age as well as fot the molecular machanism research of age and gendar associated diseases.The fourth part of the research was to systematically analyze the alterative modes, rules and possible biological functions of the potential N-glycosites in viral glycoproteins in the evolutionary process of influenza A/H1N1 viruses by employing a serial of bioinformatic tools. After predicting the potential N-glycosite in 2770 HAs and 3235 NAs of influenza A/H1N1 viruses,3D modeling of the representative glycoprotein structures and in silico glycosylation of the modeled proteins, the results showed that influenza H1N1 viruses underwent different alterations of protein glycosylation in different hosts. two alternative modes of glycosite alteration were involved in the evolution of human influenza virus:One was an increase in glycosite numbers, which mainly occurred with high frequency in the early stages of evolution. The other was a change in the positional conversion of the glycosites. which was the dominating mode with relatively low frequency in the later evolutionary stages. There are five possible biological functions for these alterations:to mask the antigenic sites. to protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. Although the results still need to be supported by experimental data, the information here may provide some constructive suggestions for research into the glycosylation of influenza viruses and even the production of viral vaccines. |
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