Hydrolytically stable hydrophilic CE coatings for protein characterization

描述(申请人提供)：该项目的主要目标是解决与毛细管电泳(CE)中使用的熔融硅管内壁涂层的当前有机膜的水解稳定性仍然不令人满意相关的分析问题。目的是通过显著降低分析物和二氧化硅表面之间的不良相互作用所造成的限制，充分利用CE的卓越效率。最终目标是获得清晰的分离轮廓，从而能够更好地表征来自各种组织或体液的复杂样品中存在的蛋白质种类。该项目将侧重于通过三步过程开发化学修饰的毛细管，该过程首先在二氧化硅表面形成无机氢化硅前体。接下来，在铂催化剂的存在下，通过氢化物中间体与末端烯烃对乙烯基氯化苄的反应来完成氯化苄基的连接。最后，在铜催化剂存在下，N-取代丙烯酰胺单体(即N-丙烯酰氨基丙醇)在氯化苄基团上引发聚合，在二氧化硅表面上化学地附着一层亲水层。因此，我们的策略结合了三个方面的优点：第一，涂层通过Si-C键非常稳定地固定在毛细管内壁上；第二，类似稳定的聚合物薄膜，其强度来自丙烯酰胺上的N-取代；以及第三，允许轻松控制涂层厚度的原位“活性”聚合方法。该项目各阶段的基本原理已在本研究人员和其他研究人员的先前工作中阐述，它们的实验可靠性已分别得到证明。修饰后的表面将通过红外光谱和固体核磁共振光谱、原子力显微镜、接触角测量等进行表征。自然，还将彻底检查一些化学探针的CE迁移曲线，最终将为选定的蛋白质组样品开发分析程序。彻底评估所有新粘结材料的稳定性也将是这项工作的重要组成部分。该项目的成功完成将对蛋白质组学、蛋白质物种的分析特征以及阐明它们在细胞功能中的生物学作用产生重大影响。 公共卫生相关性：从公共卫生的角度分析人体组织和体液中的蛋白质分子种类是极其重要的，因为了解蛋白质的组成和命运将为许多疾病的机制提供有价值的见解，并对药物开发产生重要影响。该项目将致力于开发一种改进的毛细管电泳衣，以解决与当前毛细管电泳法分离和定量蛋白质不太满意相关的分析问题。

DESCRIPTION (provided by applicant): The main objective of this project is to advance in the solution of the analytical problem associated with the still unsatisfactory hydrolytic stability of current organic films that coat the inner-wall of the fused-silica tubes used in capillary electrophoresis (CE). The purpose is to fully exploit the superb efficiency of CE by significantly decreasing the limitation due to the undesirable interactions between the analyte and the silica surface. The ultimate goal is to obtain clear-cut separation profiles that enable better characterization of the protein species present in complex samples from a variety of tissues or bodily fluids. The project will focus on the development of chemically modified capillaries through a three-step process that starts with the formation of an inorganic silicon hydride precursor on the silica surface. Next, attachment of benzyl chloride groups is accomplished by reacting the hydride intermediate with a terminal olefin, p-vinyl-benzyl chloride, in the presence of a Pt- catalyst. Finally, polymerization of an N-substituted acrylamide monomer (namely, N-acryloyl- aminopropanol), initiated at the benzyl chloride group in the presence of a Cu-catalyst, produces a hydrophilic layer chemically attached to the silica surface. Thus, our strategy brings together the best of three worlds: first, a very stable anchorage of the coating to the inner wall of the capillary tube by means of Si-C linkages; second, a similarly stable polymeric film whose strength arises from the N-substitution on acrylamide; and third, an in-situ "living" polymerization method that permits easy control of the coating thickness. The fundamentals of the various phases of the project have been set out in previous work of this researcher as well as that of others, and their experimental soundness has been separately demonstrated. The modified surfaces will be characterized by IR and solid-state NMR spectroscopies, atomic force microscopy, contact-angle measurements, etc. Naturally, the CE migration profile of a number of chemical probes will also be thoroughly examined and, eventually, analytical procedures will be developed for selected proteome samples. A thorough assessment of the stability of all new bonded materials will also be an essential part of this work. A successful completion of this project should have a significant impact on proteomics, the analytical characterization of protein species and the elucidation of their biological role in cell function. PUBLIC HEALTH RELEVANCE: Analytical mapping of protein molecular species in human tissues and fluids is extremely important from a public health point of view because understanding protein composition and fate will provide valuable insights into the mechanisms of many diseases and important implications for drug development. This project will pursue the development of an improved CE coating to move toward the solution of the analytical problem associated with the less than satisfactory separation and quantification of proteins by current CE methodologies.