| Development of selectivity separation marerials is one of the most important approaches to resolve the problems in the separation science. Aiming to the application research of saccharides separation and glycopeptides/phosphopeptides enrichment in modification-specific proteomics, a series of research work was carried out for solving the hot and difficult issues in these fields, combined with the increasing development of HPLC and MS. Based on the preparation and investigation of several novel selectivity separation materials, the specific preparation, characterization, evaluation, application in the saccharides separation and glycopeptides/phosphopeptides enrichment, and related enrichment mechanism in modification-specific proteomics were formulated systematically.Hydrophilic interaction of liquid chromatography (HILIC) is a promising alternative HPLC mode for separation of very polar compounds. A novel cellulose-coated silica HILIC stationary phase was synthesized with a method based on ionic interaction. The reulsting material was characterized by elemental analysis, N2adsorption-desorption isotherms and ξ--potential. The stationary phase was successful uesd to separate the samples of polar compounds and the complex samples of oligosaccharides, and demonstrated good reproducibility and stability. The material exhibited good separation selectivity for carbohydrates and ability to enrich glycosylated and phosphorylated peptides. Moreover, the method described here is easy to achieve, environmentally safe and innovative than other methods. It also has extensive appliction possibilites to separate other categories polar compounds.Another saccharide-based stationary phase for HILIC was presented. This method used carbonyl di-imidazole (CDI) as a cross-linker for dextran in aqueous solution. This dextran-based stationary phase was preparated with a simple method, and proved to possess good hydrophilicity and chemical stability. Sugars and sugars alcohols were well separated, with high efficiency. The separation of anomers of reducing sugars was suppressed, which is encouraging for possible applications in the quantification of carbohydrates. The separation of neutral, basic and acidic oligosaccharides was also achieved in the HILIC mode. The material has potential for usd in a HILIC-RPLC2D-LC system for the separation of complex glucoside samples in natural products. Finally, the separation material shows potential for the enrichment of glycosylated peptides, presumably better result with higher enrichment selectivity and recovery will be achieved by further optimization.System methodology was established for incestigation of the retention mechanism of phosphopepthdes enrichment in the zwitterionic material Click TE-Cys. Through the optimization of elution conditions, realizing the filally selectivity enrichment of phosphopeptides, and separated multi-phosphorylated peptides from single-phosphorlated peptides in the results as well. The optimization scheme was applied to the complex sampke system and actual sample system, this method also exhibited high selectivity for phosphopeptides enrichment in our assessment. This efficient and optimized method will be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis.Retention mechanisms between glycopeptides and TiO2were explored by checking the influence of acetonitrile content, NH3H2O concentration and formic acid concentration on the retention of glycopeptides. Both hydrophilic interaction and ligand-exchange retention mechanisms between glycopeptides and TiO2were found to be involved. Based on this, a uniform method was established for selective enrichment of glycopeptides with TiO2. The developed method exhibited high selectivity for N-linked glycopeptides with short to long peptide sequences. This efficient and uniform method will be a valuable tool for post-translational modification in proteomics. On the basis of above investigation, eight of metal doping tirania mircospheres including Ti-Al, Ti-Ce, Ti-Co, Ti-Cu, Ti-Fe, Ti-La, Ti-Mn, and Ti-Sm were prepared by sol-gel method. The obtained mixed metal oxides were characterized by HRSEX, ED AX and N2adsorption-desorption isotherms, as well, they were applied in the enrichment of phosphopeptides and glycopeptides. By examining and comparing the differences and characteristics of these mixed metal oxides in the post-translation modified-peptides enrichment, it provide a more appropriated technical for the basis of with develpment of efficient enrichment materials and enrichment methods. The data collectively indicate that the material developed in this study is of considerable value and has potential application in the fields of proteomics, which will be investigated in depth in the future. |
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