Phosphoproteomics of Oral Fluids

描述(申请人提供)：我们目前正在经历一个前所未有的生物/生物医学科学时代，我们正在列举新的、快速、灵敏和高通量的技术来研究蛋白质结构功能。其中一项技术是最先进的质谱学，它能够更全面地表征体液中的蛋白质组。唾液具有反映健康/疾病状态的潜力，因为它含有广泛的蛋白质、分析物、抗体和源自血液的激素以及腺体分泌物特有的那些。“唾液磷蛋白质组”的鉴定需要结合特定的化学方法和适用于磷肽生物化学的工具。尽管唾液中的磷蛋白含量在保护牙釉质中起着至关重要的作用，但到目前为止，磷蛋白质组学仍是唾液蛋白质组学领域中一个未被触及的领域。我们推测，来自全唾液、主要和次要腺体分泌物和牙周沟液(GCF)的口腔液体的磷蛋白质组有很大的差异。确定一般口腔液中这些成分的差异磷蛋白质组既具有高度的相关性，也是及时的，因为这样的工作结果很可能导致识别新的未知唾液磷蛋白，并在临床牙科中找到直接的应用。我们最近从健康人的全唾液中鉴定出一个大规模的磷化蛋白质组，共鉴定出114个磷化蛋白，其中~95%是迄今未知的。我们进一步假设，全唾液的磷蛋白质组与腺唾液和GCF的磷蛋白质组显著不同。事实上，我们最近的研究建立了腮腺唾液磷蛋白组，它鉴定了40种磷蛋白，仅为整个唾液磷蛋白组的1/3。这些研究第一次以提供直接证据来支持我们的概念发展和假设而告终。这与我们的假设是一致的，即腺磷蛋白质组可以在整个唾液中被磷酸酶修饰，或者额外的磷蛋白质组贡献是有效的，例如GCF。我们预计GCF将有自己独特的磷蛋白质组，因为它靠近含有丰富的细胞外基质磷蛋白的骨。在牙周病的破骨细胞的骨吸收过程中，GCF中的骨磷蛋白的存在是突出的。我们将对正常口腔健康人和牙周病患者的牙周膜样本进行磷蛋白质组研究。拟议的研究将结合我们在磷蛋白化学/生物化学方面的专业知识和最新的最先进的质谱学技术，开发和实施有效的口腔液体磷蛋白质组分析方法。这项拟议工作的成就将大大促进基于唾液的诊断/生物标志物和新疗法的未来发展，并增加另一个维度，具有相当大的健康和经济影响。 与公共健康相关：磷蛋白在维持牙齿完整性方面发挥着重要作用。随着质谱学技术的进步，现在可以建立全球范围内的磷蛋白谱及其在唾液腺中的分布。这些知识对于确定口腔环境中磷蛋白的全部保护潜力将是非常宝贵的，并有助于开发治疗口腔和/或系统性人类疾病的新的治疗方法。

DESCRIPTION (provided by applicant): We are currently experiencing an unprecedented times in biological/biomedical sciences with enumerate new, rapid, sensitive and high throughput technologies to study protein-structure function. One of these technologies is state-of-the-art mass spectrometry which has the capacity to characterize more globally the proteomes in body fluids. Saliva has the potential to reflect a measure of health/disease state, since it contains a wide range of proteins, analytes, antibodies and hormones derived from blood as well as those specific to glandular secretions. The identification of the "phosphoproteome of saliva" requires the use of combination of specific chemistries and tools applicable to phosphopeptide biochemistry. Phosphoproteomics to date is an untouched area in the saliva proteomics field, despite the fact that it is the phosphoprotein contents of saliva that have been demonstrated to be of vital importance in the protection of tooth enamel. We hypothesize that there are major differences in the phosphoproteome of oral fluids derived from whole saliva, major and minor glandular secretions and gingival crevicular fluid (GCF). It is both highly relevant and timely to define the differential phosphoproteome of each of these contributors to the general oral fluid since results of such work will most likely lead to the identification of novel unknown salivary phosphoproteins, and find direct applications in clinical dentistry. Our recent work with whole saliva from healthy individuals led to identification of a large-scale phosphoproteome of this fluid with 114 phosphoproteins identified of which ~95% were hitherto unknown. We further hypothesize that the phosphoproteome of whole saliva differs significantly from those of glandular salivary secretions and GCF. Indeed, our most recent study established the parotid saliva phosphoproteome which identified 40 phosphoproteins, only ~1/3 that of the whole saliva phosphoproteome. These studies for the first time culminated in providing direct evidence in support of our conceptual developments and hypothesis. This is consistent with our hypothesis that glandular phosphoproteome can be modified in whole saliva by e.g., phosphatases, or that additional phosphoproteome contributions are in effect, e.g., GCF. We expect that GCF will have its own distinct phosphoproteome due to its close proximity to the bone which contains abundant extracellular matrix phosphoproteins. The presence of bone phosphoproteins in GCF is eminent during bone resorption by osteoclasts in periodontal disease. We will investigate the phosphoproteome of GCF samples from subjects with normal oral health and those with periodontal disease. The proposed studies will develop and implement efficient approaches to the phosphoproteome analysis of oral fluids by combining our expertise in phosphoprotein chemistry/biochemistry interfaced with the latest state-of-the-art mass spectrometric technology. The accomplishments of the proposed work herein will contribute significantly and add another dimension to the future development of saliva based diagnostics/biomarkers and new therapeutics with considerable health and economic impact. PUBLIC HEALTH RELEVANCE: Phosphoproteins fulfill an essential role in the maintenance of the integrity of teeth. With the advances in mass spectrometric techniques, a global spectrum of phosphoproteins and their distribution amongst the salivary glands can now be established. Such knowledge will be invaluable in determining the full protective potential of phosphoproteins in an oral environment and help to develop novel therapeutic approaches for the treatment of oral and/or systemic human diseases.