PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。获得性牙釉质膜是主要来源于唾液成分的有机膜，其覆盖并保护牙齿的牙釉质表面。该项目的最终目的是表征获得的釉质表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，经典的蛋白质分离色谱法，抗体检测已受到限制的材料量小。这些方法已经表明，表膜由难以与表膜整体相关的量的一种组分或另一种组分组成。过去的努力，特别是使用放射性标记的蛋白质和质谱分析的薄膜蛋白质分离的二维电泳的吸附研究，提供了信息，表明磷酸化蛋白质的含量高。此外，富含脯氨酸的蛋白质，唾液酸甘油酶和半胱氨酸蛋白酶抑制剂SN已被确定在人工薄膜中产生的唾液分泌物吸附到羟基磷灰石。然而，当对来自体内收集的表膜的蛋白质进行类似的分析时，富含脯氨酸的蛋白质明显不存在。通过分析提取的胰蛋白酶肽，对来自唾液和体内膜的2D凝胶点进行表征。使用使用MALDI-TOF和Q-oTOF获得的MS和MS/MS数据的数据库检索鉴定了预期的唾液蛋白（半胱氨酸蛋白酶抑制剂、淀粉酶、唾液酸甘油酶等）以及非预期的牙龈血清液蛋白（即，钙颗粒蛋白）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化学杂志上。为了提供唾液和薄膜成分的更全面的图片，现在正在采用离子交换色谱法结合在线毛细管LC/MS。为了便于这种分析，已经构建了一个定义的数据库，其中包含所有已知的口腔蛋白质和其他可能存在于口腔中的组分。在capLC-QStar i nanoESI-正交TOF MS系统的智能数据采集（IDA）操作期间采集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。使用这种方法显著增加了洗脱峰的归属，并产生了高度可靠和一致的相关结果。目前正在编写一份介绍这些结果的手稿。这种方法将导致深入了解蛋白质合成/降解途径和功能，在口腔液和人类pellic

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