Hydrolytically stable hydrophilic CE coatings for protein characterization

描述（由申请人提供）：该项目的主要目标是推进与毛细管电泳（CE）中使用的覆盖在熔融硅管内壁上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的解决。目的是通过显著降低由于分析物与二氧化硅表面之间的不良相互作用而产生的限制，充分利用CE的卓越效率。最终目标是获得清晰的分离谱，以便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工艺开发化学修饰的毛细管，首先是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反应，实现氯化苄基团的连接。最后，n取代丙烯酰胺单体（即n -丙烯酰-氨基丙醇）在cu催化剂的存在下，在氯化苄基上引发聚合，产生一层化学附着在二氧化硅表面的亲水层。因此，我们的策略将三个方面的优点结合在一起：首先，通过Si-C连接将涂层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；第三，一种原位“活”聚合方法，可以很容易地控制涂层厚度。该项目各个阶段的基本原理已经在本研究人员以及其他研究人员之前的工作中提出，并且它们的实验合理性已经分别得到了证明。改性后的表面将通过红外光谱、固体核磁共振光谱、原子力显微镜、接触角测量等进行表征。当然，许多化学探针的CE迁移剖面也将被彻底检查，最终，将为选定的蛋白质组样品开发分析程序。对所有新粘结材料的稳定性进行全面评估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、蛋白质物种的分析表征及其在细胞功能中的生物学作用的阐明产生重大影响。

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.