Top-Down Mass Spectrometry of Salivary Fluids for Cancer Assessment

描述（由申请人提供）： 将进行自上而下的蛋白质组分析和蛋白质表征，以可重复地测量人全唾液中的1500种蛋白质特征，以评估唾液用于癌症检测和诊断的适用性。血浆或血液已被公认为用于测量蛋白质和其他生物分子水平以进行临床测试的生物流体的选择，但对于其他生物介质可能存在优势，其可以更容易地获得以用于临床采样。唾液中含有丰富的蛋白质组，这些蛋白质组来源于唾液腺、口腔内壁和血液。唾液很容易获得，与其他体液相比更容易收集。与血浆相比，具有高度丰富的白蛋白，转铁蛋白和IgG，唾液蛋白质组对获得整个蛋白质补体的挑战较少。出于这些原因，使用唾液进行癌症诊断是一个有吸引力的选择。蛋白质预分离策略将被开发用于划分唾液蛋白质组，以通过自上而下的蛋白质组学进行进一步表征。将研究三种方法：通过HPLC通过其疏水性分离蛋白质;溶液等电聚焦以基于其pI将复杂混合物划分为5-7个单独的子样品;通过酰肼树脂方法亲和选择N-连接的糖蛋白，并且将释放先前糖基化的蛋白质用于随后的MS分析。目标将是分辨和观察比没有预分级分离的情况下更多的低丰度蛋白质。一种基于激光解吸和电喷雾电离相结合的新方法，称为电喷雾辅助激光解吸/电离（ELDI），将进一步开发，以提供自上而下的MS/MS生成的序列标签，以识别癌症的相关蛋白质标志物。蛋白质预分级分离和MALDI/ELDI-MS自上而下蛋白质组学的整体平台将应用于来自健康对照和口腔癌受试者的唾液样品。目标是在健康受试者的唾液样本中可重复地识别1500种蛋白质特征，并将这些特征与口腔癌样本中测量的唾液蛋白质进行比较。与公共卫生的相关性：唾液可能是识别癌症等疾病标志物的有吸引力的样本。自上而下的蛋白质组学将用于比较健康个体和癌症受试者唾液中的蛋白质。

DESCRIPTION (provided by applicant): Top-down proteome profiling and protein characterization to measure reproducibly 1500 protein features in human whole salivary fluids will be performed to assess the suitability of saliva for cancer detection and diagnosis. Plasma or blood has been the accepted biofluid of choice for measuring levels of proteins and other biomolecules for clinical testing, but there may be advantages for other biomedia which can be more readily obtained for clinical sampling. Saliva has a rich proteome that is derived from the salivary glands, the linings of the oral cavity, and blood. Saliva is readily available and easier to collect compared to other body fluids. Compared to plasma, with highly abundant albumin, transferrin, and IgGs, the salivary proteome presents fewer challenges for accessing the entire protein complement. For these reasons, the use of saliva for cancer diagnostic purposes presents an attractive option. Protein pre-fractionation strategies will be developed to partition the salivary proteome for further characterization by top-down proteomics. Three methods will be investigated: separation of proteins by their hydrophobicity via HPLC; solution isoelectric focusing to partition the complex mixture into 5-7 separate sub-samples on the basis of their pl; affinity selection of N-linked glycoproteins by a hydrazide resin method, and the formerly glycosylated proteins will be released for subsequent MS analysis. The goal will be to resolve and observe more lower abundance proteins than without pre-fractionation. A new method based on a combined laser desorption and electrospray ionization, termed electrospray-assisted laser desorption/ionization (ELDI), will be further developed to provide top-down MS/MS-generated sequence tags to identify the relevant protein markers of cancer. The overall platform of protein pre-fractionation and MALDI/ELDI-MS top-down proteomics will be applied to saliva samples from healthy control and oral cancer subjects. The goal will be to identify 1500 protein features reproducibly in the saliva samples from the healthy subjects and compare these characteristics to the saliva proteins measured in oral cancer samples. Relevance to public health: Salivary fluid may be an attractive sample for the identification of markers of diseases, such as cancer. Top-down proteomics will be applied to compare proteins in saliva from healthy individuals and cancer subjects.