PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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，抗体检测受到限制的材料量小。这些方法表明，表膜由一种成分或另一种成分组成，其量难以与表膜整体相关。 过去的努力，特别是吸附研究，使用放射性标记的蛋白质和质谱分析的薄膜蛋白分离的2-D PAGE，提供的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，statherin和半胱氨酸蛋白酶抑制剂SN，被确定在由唾液分泌物吸附到羟基磷灰石产生的人工薄膜。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MALDI-TOF和Q-oTOF MS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）。我们的研究结果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，采用了离子交换色谱法结合在线毛细管LC/MS。为了促进这一分析，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口腔中可能存在的其他组分，并且正在查阅唾液蛋白质组研究的结果。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可用数据库快速搜索，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。 这种方法应导致深入了解蛋白质的合成/降解途径和功能，在口腔液和人类薄膜。Oppenheim教授的部门最近收购了自己的LTQ MS，以便将更多时间用于这些分析。顾问人员将继续提供咨询和意见。

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 were limited by the small amounts of material. These methods 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-D PAGE, provided information indicating a high content of phosphorylated proteins. Additionally, proline-rich proteins, statherin and cystatin SN, were 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 MS 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 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 has been 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 and the resuls from the salivary proeome study are being consulted. Data acquired during intelligent data acquisition (IDA) operation of the capLC-QStar i nanoESI-orthogonal TOF MS system can be quickly searched against the available databases, and searches that allow for all known post-translational 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. This approach should lead to an in-depth understanding of protein synthesis/degradation pathways and functions in oral fluids and human pellicle. Prof. Oppenheim's department recently acquired its own LTQ MS in order to dedicate more time to these analyses. Resource personnel will continue to provide advice and consultation.