PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

口腔液和牙膜层中的蛋白质

• 批准号: 7722973
• 负责人: Frank G Oppenheim
• 金额: $ 0.07万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722973
• 关键词: Adsorption; Amylases; Antibodies; Blood capillaries; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Consult; Consultations; Coupled; Cystatins; Data; Databases; Degradation Pathway; Dental Enamel; Dental Pellicle; Depth; Detection; Film; Funding; Gel; Gingiva; Grant; Human; Human Resources; Hydroxyapatites; Institution; Ion-Exchange Chromatography Procedure; Keratin; Lead; Leukocyte L1 Antigen Complex; Liquid substance; Methods; Operative Surgical Procedures; Oral; Oral cavity; Peptides; Post-Translational Protein Processing; Property; Protein Analysis; Protein Biosynthesis; Proteins; Publishing; Radiolabeled; Research; Research Personnel; Resources; Saliva; Salivary; Salivary Proteins; Serum; Source; Spottings; System; Time; Tooth structure; United States National Institutes of Health; capillary; cystatin SN; data acquisition; gel electrophoresis; in vivo; liquid chromatography mass spectrometry; proline-rich proteins; radiotracer; saliva secretion; statherin; tooth surface

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 后天的牙釉质薄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。 该项目的最终目标是表征所获得的牙釉质薄膜的成分和功能特性。 先前的研究使用凝胶电泳、经典蛋白质分离色谱和抗体检测等方法，但由于材料量少而受到限制。这些方法表明，薄膜由一种或另一种组分组成，其数量难以与薄膜整体相关。 过去的努力，特别是使用放射性标记蛋白质的吸附研究和通过 2-D PAGE 分离的薄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，在唾液分泌物吸附到羟基磷灰石上产生的人造薄膜中还发现了富含脯氨酸的蛋白质，富酪蛋白和半胱氨酸蛋白酶抑制剂SN。然而，当对来自体内收集的薄膜的蛋白质进行类似分析时，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来表征唾液和体内表膜的 2D 凝胶点。 使用 MALDI-TOF 和 Q-oTOF MS 获得的 MS 和 MS/MS 数据进行数据库搜索，识别出预期的唾液蛋白（半胱氨酸蛋白酶抑制剂、淀粉酶、富酪蛋白等），以及非预期的牙龈血清蛋白（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发表在 J. Biol 上。化学。 为了提供唾液和薄膜成分的更全面的图像，采用了离子交换色谱法与在线毛细管 LC/MS 相结合。为了促进这一分析，已经构建了一个包含所有已知口腔蛋白质和口腔中可能存在的其他成分的明确数据库，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) 操作过程中获取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。使用这种方法显着增加了洗脱峰的分配，并产生了高度可靠且一致相关的结果。 这种方法应该有助于深入了解口腔液和人体表膜中的蛋白质合成/降解途径和功能。 Oppenheim 教授的部门最近购买了自己的 LTQ MS，以便将更多时间用于这些分析。资源人员将继续提供建议和咨询。