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图谱和蛋白质特征分析，以重复性地测量人类全唾液中的1500个蛋白质特征，以评估唾液是否适合于癌症检测和诊断。血浆或血液一直是临床测试中测量蛋白质和其他生物分子水平的公认生物流体，但其他生物介质可能也有优势，可以更容易地获得临床采样。唾液含有丰富的蛋白质组，来源于唾液腺、口腔衬里和血液。与其他体液相比，唾液很容易获得，也更容易收集。与具有高度丰富的白蛋白、转铁蛋白和免疫球蛋白的血浆相比，唾液蛋白质组对获得整个蛋白质补体的挑战更小。出于这些原因，将唾液用于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来划分唾液蛋白质组，以供自上而下的蛋白质组学进一步表征。将研究三种方法：通过高效液相分离蛋白质的疏水性；溶液等电聚焦根据其PL将复杂混合物划分为5-7个单独的亚样品；通过肼树脂方法亲和选择N-连接的糖蛋白，并将先前糖基化的蛋白质释放用于后续的MS分析。我们的目标将是比没有分离前更多地分解和观察到更多的低丰度蛋白质。将进一步开发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)，以提供自上而下的MS/MS生成的序列标签来识别癌症的相关蛋白质标记物。蛋白质预分离和MALDI/ELDI-MS自上而下蛋白质组学的整体平台将应用于健康对照和口腔癌受试者的唾液样本。目标将是在健康受试者的唾液样本中识别1500种重复的蛋白质特征，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。与公众健康相关：唾液可能是识别癌症等疾病标志物的一个有吸引力的样本。自上而下的蛋白质组学将应用于比较健康人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.