PROTEINS IN THE ORAL FLUIDS AND TOOTH PELLICLE LAYER

口腔液和牙膜层中的蛋白质

• 批准号: 7601967
• 负责人: Frank G Oppenheim
• 金额: $ 0.11万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601967
• 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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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、抗体检测法等方法，但由于所用材料较少，限制了以往的研究。这些方法表明，膜是由一种或另一种成分组成的，其数量很难与膜作为一个整体联系起来。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2-D PAGE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产生的人工膜中发现了富含Pro的蛋白质，Statherin和Cystatin 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。利用MALDI-TOF和Q-OTOF MS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、他汀类药物和其他)，以及意想不到的牙周血清液体蛋白(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，采用了离子交换层析与在线毛细管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可能存在于口腔中的其他成分，并正在参考唾液益生体研究的结果。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可用的数据库快速搜索，并且可以快速和可靠地执行允许所有已知的翻译后修饰的搜索。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。这种方法应该导致对口腔液和人体膜中蛋白质合成/降解途径和功能的深入了解。奥本海姆教授的系最近收购了自己的LTQ MS，以便投入更多时间进行这些分析。资源人员将继续提供咨询和咨询。