钛胶整体HPLC柱的制备及其在磷酸蛋白质组学分析中的应用

项目名称： 钛胶整体HPLC柱的制备及其在磷酸蛋白质组学分析中的应用

项目编号： No.20875069

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 姜子涛

作者单位： 天津商业大学

项目金额： 30万元

中文摘要： 利用Sol-gel技术合成了钛胶基质HPLC整体柱，探讨了影响合成钛胶基质整体柱的各项因素，如反应温度、络合剂、反应溶剂、加水量、催化剂、反应及凝胶化时间等。通过控制反应条件，优化了钛胶基质整体柱的比表面、孔结构和骨架。利用XRD、SEM、BET等手段对所合成的钛胶整体柱进行了表征，利用硅烷化技术合成了钛胶反相固定相，提出钛胶基质整体柱制备的新方法和新技术。测试了所合成的钛胶整体固定相的色谱性能和色谱行为。并在此基础上利用钛胶固定相HPLC法，采用亲和色谱和RP-HPLC的方法，应用于食品及生物样品中大豆卵磷脂、维生素B、三聚氰胺和三聚氰酸、苯甲酸、香兰素、叶酸、咖啡因、烟酰胺、柠檬酸、阿斯巴甜等的测定了取得了令人满意的结果。同样地，胰蛋白酶促水解得到的酪蛋白水解肽混合物在钛胶固定相上也得到了较好的分离。钛胶整体固定相对克服硅胶和聚合物固定相的不足，扩大整体柱的应用范围有积极的作用。结果显示，钛胶固定相由于不存在硅胶固定相的"第二效应"的弊端，以及能够选择性地吸附磷酸肽，必将在蛋白质和肽类化合物、碱性化合物、羧酸类化合物、氨基酸等分离分析，以及蛋白质（肽）组学研究方面发挥出巨大作用。

中文关键词： 钛胶基质整体柱；HPLC；固定相；分离

英文摘要： Using sol-gel technology, titania monolith stationary phase for HPLC was synthesized. The influence of factors such as reaction temperature, reaction solvent, complexing agent, water addition, catalyst, reaction time and gel time on synthesis of titania monolith were studied. The surface area, pore structure and framework of titania monolith were optimized through controlling the reaction conditions. The synthesized titania monoliths were characterized by XRD, SEM, and BET methods. Reverse-phase titania monolith stationary phase was synthesized by use of silane technique. A new method for the preparation of titania monolith column has been developed. The chromatographic performance and chromatographic behaviors of synthesized titania monolithic stationary phase were determined. The basis of titania-based stationary phase for HPLC, soy lecithin, vitamin B, melamine and melamic acid, benzoic acid, vanillin, folic acid, caffeine, niacinamide, citric acid, aspartame in the food and biological samples were determined using affinity chromatography and RP-HPLC method with satisfactory results. Likewise, the casein peptides mixture obtained by trypsin hydrolysis was also separated much better in titania monolith. Titania overcomes the deficiency of silica and polymer stationary phase and expands the application range of the monolith with a positive effect. The results show that titania stationary phase because it does not exist in the silica the disadvantages of "the second effect", and can selectively adsorb phosphoric peptide, it will play a important role in separation and analysis of the proteins and peptides, alkalines, carboxylic acids, amino acids as well as proteome (peptide) research.

英文关键词： Titania monolithic column; HPLC; stationary phase; separation