Tissue Lysates for Studies of Protein Phosphorylation

用于蛋白质磷酸化研究的组织裂解物

• 批准号: 7503182
• 负责人: BEATRICE S KNUDSEN
• 金额: $ 23.76万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-08 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7503182
• 关键词: Alcohols; Biological Assay; Biological Markers; Biological Preservation; Biopsy; Buffers; Cancer Patient; Classification; Clinical Trials; Community Hospitals; Condition; Development; Diamond; Dot Immunoblotting; Drug Delivery Systems; End Point; Epidermal Growth Factor Receptor; Evaluation; Fixatives; Formalin; Freezing; Goals; Grant; Human; MAP Kinase Gene; Malignant Neoplasms; Measurement; Measures; Methods; Patients; Performance; Pharmaceutical Preparations; Phosphoproteins; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Preparation; Procedures; Process; Proteins; Protocols documentation; Public Health; Rate; STAT3 gene; Sampling; Signal Pathway; Signal Transduction; Solid Neoplasm; Spottings; Staining method; Stains; Statistical Methods; Stratification; Testing; Threonine; Tissue Fixation; Tissue Procurements; Tissue Sample; Tissues; Tyrosine; Validation; Xenograft procedure; base; cancer classification; clinically relevant; clinically significant; crosslink; improved; kinase inhibitor; novel; outcome forecast; phosphatase inhibitor; prognostic; response; sample fixation; tissue fixing; tissue preparation; tissue processing

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.

描述（由申请人提供）：本项目的长期目标是测量人类癌症组织中的磷蛋白。作为实现这一目标的第一步，该R21赠款的目的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制备组织样品的最佳条件。将异种移植组织冷冻或固定在非交联固定剂或福尔马林中。用4种缓冲液提取蛋白质，并使用斑点印迹测定法分析蛋白质磷酸化。在目标1中，我们测量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标或预测生物标志物具有临床意义的蛋白质中的特异性磷酸化位点。我们使用统计学方法来确定样品制备方案，该方案是（1）可重复的，（2）提供高产量的提取蛋白质，和（3）在提取过程中保持蛋白质磷酸化。我们对结果进行排名，并将排名最高的方案与参考样品制备程序进行比较。我们预计，通过确定用于测量癌症组织中全局和特异性蛋白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。 公共卫生相关性声明：磷蛋白代表了人类癌症中有效的预测和预后生物标志物;然而，它们的表达不稳定，并且它们的测量困难。该项目的目的是测试新的样品制备方案，可以更好地保留人类癌症组织中的蛋白质磷酸化，以进行精确和可重复的测量。