【作者】 盛骞莹；

【导师】 梁鑫淼； 柯燕雄；

【作者基本信息】 华东理工大学 ， 制药工程与技术， 2014， 博士

【摘要】 本论文基于对糖分离及修饰蛋白质组学中糖肽／磷酸化肽富集的选择性分离材料的应用与研究,结合日益发展的色谱、质谱等手段,针对目前糖类化合物分离及翻译后修饰蛋白质组学研究中面临的热点难点问题展开了一系列研究工作。通过制备与考察几种新型的选择性分离材料,对其制备、表征、分离评价、及在糖分离、糖肽／磷酸化肽选择性富集中的应用以及相关富集分离机理做了系统地阐述与研究。利用静电作用制备了一种新型的涂覆纤维素糖固载亲水色谱材料,通过元素分析、氮气吸附脱附实验及ζ-电势测试对材料的固载量、孔结构及表面带电情况的进行了系统的表征。以核苷碱基和糖类化合物等极性物质为模型化合物进行了色谱评价,结果表明该新型固定相具有典型的亲水作用保留特性,适合用于分离小分子糖、寡糖样品及天然产物中的糖苷等各类强极性化合物,将该固定相材料应用于翻译后修饰肽的富集也表现出很好糖肽／磷酸化肽选择性富集效果。利用羰基二咪唑为反应连接臂将葡聚糖键合到硅胶上,制备了键合多糖型亲水色谱材料。该方法创新地将葡聚糖与硅胶进行结合,可用于糖类化合物特异性的分离与糖肽选择性富集。键合葡聚糖亲水色谱固定相材料在糖、糖醇样品,高聚合度的寡糖样品与天然产物中的糖苷样品的分离及糖蛋白酶解液样品糖肽的富集都表现出很好的应用能力。将该制备方法进行推广,制备了其他几种多糖或多羟基固载硅胶固定相,为新型亲水色谱固定相材料的发展提供了更为广阔的发展空间。考察了两性离子材料Click TE-Cys对磷酸化肽的富集作用机理,通过对上样和淋洗条件的优化,实现该材料对磷酸化肽的高选择性富集,同时很好地将单磷酸化肽和多磷酸化肽进行分段富集。特别值得一提的Click TE-Cys对多磷酸化肽表现出优异的富集选择性。所建立的方法对于复杂样品体系及实际样品体系都表现出较好的磷酸化肽富集效果。该方法的建立对磷酸化修饰蛋白质组学的研究有着积极的意义。考察了氧化钛对糖肽的富集作用机理,进而发展了一种简单统一的糖肽富集方法。在此基础上,发展了复合金属氧化物材料,利用HRSEX、EDAX、氮气吸附脱附等对其球形形貌及比表面等参数进行了系统表征,表明通过溶胶-凝胶法制备的复合金属氧化物材料是一类理想的富集翻译后修饰肽的分离材料。通过考察和比较各种复合金属氧化物材料在磷酸化肽及糖肽的选择性富集中的差异和特性,以提供更为合适的磷酸化肽／糖肽富集手段,为发展高效的翻译后修饰肽富集材料及富集方法提供了基础。更多还原

【Abstract】 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.更多还原