Tissue Lysates for Studies of Protein Phosphorylation

描述（由申请人提供）：该项目的长期目标是测量人类癌症组织中的磷酸化蛋白。作为实现这一目标的第一步，本R21基金的目标是通过测试组织固定和蛋白质提取缓冲液的多种组合来确定组织样品制备的最佳条件。异种移植物组织被冷冻或固定在非交联固定物或福尔马林中。用4个缓冲液提取蛋白质，并使用点印迹法分析蛋白质磷酸化。在Aim 1中，我们测量了酪氨酸和苏氨酸的全局磷酸化。我们还使用proQ Diamond检测所有磷酸化蛋白。在目标2中，我们评估了作为药物靶点或预测性生物标志物具有临床意义的蛋白质中的特定磷酸化位点。我们使用统计方法来确定样品制备方案：(1)可重复性，(2)提供高产量的提取蛋白质，(3)在提取过程中保存蛋白质磷酸化。我们对结果进行排序，并将排名靠前的方案与参考样品制备程序进行比较。我们期望通过确定用于测量癌症组织中整体和特定蛋白质磷酸化的样品制备方案，我们将改善许多实体瘤患者的药物选择和反应率。

DESCRIPTION (provided by applicant): The long-term objective of this project is to measure phosphoproteins in human cancer tissues. As the first step towards this goal, the objective of this R21 grant is to identify optimal conditions for preparation of tissue samples by testing multiple combinations of tissue fixation and protein extraction buffers. Xenograft tissues are frozen or fixed in a non-crosslinking fixative or in formalin. Proteins are extracted with 4 buffers and analyzed for protein phosphorylation using a dot-blot assay. In Aim 1, we measure global phosphorylation on tyrosine and threonine. We also use proQ Diamond to detect all phosphorylated proteins. In Aim 2 we evaluate specific phosphorylation sites in proteins that are of clinical significance as drug targets or predictive biomarkers. We use statistical methods to identify sample preparation protocols that are (1) reproducible, (2) provide a high yield of extracted proteins, and (3) preserve protein phosphorylation during the extraction process. We rank the results and compare the top ranked protocols to a reference sample preparation procedure. We anticipate that by identifying sample preparation protocols for measurement of global and specific protein phosphorylation in cancer tissues, we will improve drug selection and response rates for many patients with solid tumors. Public Health Relevance Statement: Phosphoproteins represent effective predictive and prognostic biomarkers in human cancers; however, their expression is unstable and their measurement difficult. The objective of this project is to test novel sample preparation protocols that may better preserve protein phosphorylation in human cancer tissues for precise and reproducible measurement.