PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。获得性牙釉质膜是主要来源于唾液成分的有机膜，覆盖和保护牙齿的牙釉质表面。本项目的最终目的是表征获得的搪瓷膜的组成和功能特性。以前的研究，使用的方法包括凝胶电泳、经典蛋白质分离色谱和抗体检测，都受到材料数量少的限制。这些方法表明，膜是由一种成分或另一种成分组成的，其数量难以与整个膜相关联。过去的努力，特别是利用放射性标记蛋白质的吸附研究和用二维电泳分离的膜膜蛋白质的质谱分析，提供了表明磷酸化蛋白质含量高的信息。此外，在唾液分泌物与羟基磷灰石吸附产生的人工膜中，还发现了富含脯氨酸的蛋白质、施他汀蛋白和胱抑素SN。然而，当对来自体内收集的细胞膜的蛋白质进行类似的分析时，富含脯氨酸的蛋白质明显缺失。通过分析提取的色氨酸肽，对唾液和体内细胞膜的二维凝胶斑点进行表征。使用MALDI-TOF和Q-oTOF获得的质谱和质谱/质谱数据进行数据库搜索，确定了预期的唾液蛋白（胱他汀类，淀粉酶，他汀蛋白等），以及意想不到的牙龈血清液蛋白（即钙粒蛋白）和细胞蛋白（内皮角蛋白）。我们的研究结果最近发表在《J. Biol》杂志上。化学。为了提供更全面的唾液和细胞膜成分的图像，离子交换色谱联用在线毛细管LC/MS正在被采用。为了便于分析，已经构建了一个包含所有已知口腔蛋白质和其他可能存在于口腔中的成分的定义数据库。在capLC-QStar i纳米esi -正交TOF质谱系统的智能数据采集（IDA）操作过程中获得的数据可以根据该数据库快速搜索，并且可以快速可靠地执行所有已知的翻译后修饰的搜索。使用这种方法大大增加了洗脱峰的分配，并产生了高度可靠和一致的相关结果。描述这些结果的手稿正在准备中。这种方法将导致对口服液和人体肾盂中蛋白质合成/降解途径和功能的深入了解

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The acquired enamel pellicle is the organic film primarily derived from salivary components that covers and protects the enamel surface of teeth. The ultimate aim of this project is to characterize the composition and functional properties of the acquired enamel pellicle. Previous studies, using methods including gel electrophoresis, classical protein separation chromatography, and antibody detection have been limited by the small amounts of material. These methods have suggested that the pellicle is composed of one component or another in amounts difficult to relate to the pellicle as a whole. Past efforts, in particular adsorption studies using radiolabelled proteins and mass spectral analysis of pellicle proteins separated by 2-dimensional electrophoresis, have provided information indicating a high content of phosphorylated proteins. Additionally, proline-rich proteins, statherin and cystatin SN have been identified in artificial pellicles produced by adsorption of salivary secretions to hydroxyapatite. However, when a similar analysis of proteins derived from in vivo collected pellicle was conducted, the proline-rich proteins were notably absent. Characterization of 2D gel spots from saliva and in vivo pellicle was performed by analyzing extracted tryptic peptides. Database searches using both MS and MS/MS data obtained using MALDI-TOF and Q-oTOF identified expected salivary proteins (cystatins, amylase, statherin, and others), as well as unanticipated gingival serum fluid proteins (i.e., calgranulin) and cellular proteins (endothelial keratins). Our results from this study were recently published in J. Biol. Chem. In order to provide an even more comprehensive picture of salivary and pellicle components, ion exchange chromatography coupled with on-line capillary LC/MS is now being employed. To facilitate this analysis, a defined database containing all the known oral proteins and other components that can be present in the oral cavity has been constructed. Data acquired during intelligent data acquisition (IDA) operation of the capLC-QStar i nanoESI-orthogonal TOF MS system can be quickly searched against this database, and searches that allow for all known posttranslational modifications can be performed rapidly and reliably. Use of this approach has significantly increased the assignments of eluting peaks and has yielded highly reliable and consistently relevant results. A manuscript describing these results is being prepared. This approach should lead to an in-depth understanding of protein synthesis/degradation pathways and functions in oral fluids and human pellic