Phosphoproteomics of Oral Fluids

描述（由申请人提供）：我们目前正在经历一个前所未有的时代，在生物/生物医学科学与枚举新的，快速的，灵敏的和高通量的技术来研究蛋白质结构功能。这些技术之一是最先进的质谱法，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/疾病状态的测量的潜力，因为它包含来自血液的广泛的蛋白质、分析物、抗体和激素以及对腺体分泌物特异的那些。“唾液磷酸化蛋白质组”的鉴定需要使用适用于磷酸化肽生物化学的特定化学和工具的组合。磷酸化蛋白质组学迄今为止是唾液蛋白质组学领域的一个未触及的领域，尽管事实上，唾液中的磷酸化蛋白含量已被证明在保护牙釉质中至关重要。我们推测，有来自全唾液，主要和次要腺体分泌物和龈沟液（GCF）的口腔液中的磷酸化蛋白质组的主要差异。这是高度相关的和及时的，以确定每个这些贡献者的差异磷酸化蛋白质组的一般口腔液，因为这样的工作的结果将最有可能导致新的未知的唾液磷蛋白的鉴定，并发现在临床牙科的直接应用。我们最近对健康个体的全唾液进行的研究鉴定了这种液体的大规模磷酸化蛋白质组，鉴定了114种磷酸化蛋白质，其中约95%是迄今为止未知的。我们进一步假设，磷酸化蛋白质组的整个唾液显着不同的腺唾液分泌物和GCF。事实上，我们最近的研究建立了腮腺唾液磷酸化蛋白质组，鉴定了40种磷酸化蛋白质，仅为整个唾液磷酸化蛋白质组的约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。这与我们的假设一致，即腺磷酸化蛋白质组可以在全唾液中被修饰，磷酸酶，或者额外的磷酸蛋白质组贡献有效，例如，全球合作框架。我们预计，GCF将有自己独特的磷酸化蛋白质组，由于其接近骨，其中含有丰富的细胞外基质磷蛋白。骨磷蛋白在牙周病破骨细胞的骨吸收过程中起重要作用。我们将调查磷酸化蛋白质组的GCF样本，从正常的口腔健康和牙周疾病。 拟议的研究将通过结合我们在磷蛋白化学/生物化学方面的专业知识与最新的最先进的质谱技术，开发和实施有效的口腔液磷酸化蛋白质组分析方法。本文提出的工作的成就将对基于唾液的诊断/生物标志物和具有相当大的健康和经济影响的新疗法的未来发展做出重大贡献并增加另一个维度。 公共卫生相关性：磷蛋白在维持牙齿完整性方面发挥着重要作用。随着质谱技术的进步，现在可以建立磷蛋白的全球谱及其在唾液腺中的分布。这些知识在确定磷蛋白在口腔环境中的全部保护潜力方面将是非常宝贵的，并有助于开发用于治疗口腔和/或全身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.