PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。获得的釉质膜是一种有机薄膜，主要来自唾液成分，覆盖和保护牙齿的釉质表面。该项目的最终目标是表征获得的釉质膜的组成和功能特性。以往的研究主要采用凝胶电泳法、经典的蛋白质分离层析法和抗体检测等方法，但由于材料的数量较少，限制了以往的研究。这些方法表明，薄膜是由一种或另一种成分组成的，其数量很难与薄膜整体相关。过去的工作，特别是使用放射性标记蛋白质的吸附研究和用二维电泳法分离的膜蛋白质的质谱分析，提供了表明磷酸化蛋白质含量很高的信息。此外，在唾液分泌物吸附到羟基磷灰石上所产生的人工膜中也发现了富含脯氨酸的蛋白质、施他林和胱抑素SN。然而，当对来自活体收集的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。通过分析提取的胰蛋白酶多肽，对唾液和体内膜的2D凝胶斑点进行了表征。使用MALDI-TOF和Q-OTOF获得的MS和MS/MS数据进行数据库搜索，确定了预期的唾液蛋白(半胱氨酸氨基转移酶、淀粉酶、他汀类蛋白和其他)，以及意外的牙周血清液蛋白(即钙粒蛋白)和细胞蛋白(内皮角蛋白)。我们这项研究的结果最近发表在《生物》杂志上。化学。为了提供更全面的唾液和膜层成分的图像，现在正在使用离子交换色谱与在线毛细管LC/MS联用。为了促进这一分析，已经建立了一个定义的数据库，其中包含所有已知的口腔蛋白和其他可能存在于口腔中的成分。在capLC-QStar I纳米ESI-正交TOF MS系统的智能数据采集(IDA)操作期间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允许所有已知翻译后修饰的搜索。这种方法的使用大大增加了洗脱峰的指定值，并产生了高度可靠和一致相关的结果。描述这些结果的手稿正在准备中。这种方法应该导致对口腔液和人体骨盆中蛋白质合成/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