Molecular screening: MALDI-TOF scanning of peptides (RMI)

分子筛选：肽的 MALDI-TOF 扫描 (RMI)

• 批准号: 7493072
• 负责人: Stephen J. Kron
• 金额: $ 54.77万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7493072
• 关键词: Acrylamide; Acrylamides; Agonist; Algorithms; Antibodies; Arts; Biological Assay; Cell Cycle; Cell Extracts; Cell Line; Cells; Cessation of life; Characteristics; Chemicals; Chemistry; Class; Clinic; Code; Compatible; Computer software; Condition; Cytolysis; Detection; Disease; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Enzymes; Epidermal Growth Factor Receptor; Gefitinib; Goals; Growth; Hydrogels; Imatinib; Immunologics; Incubated; Individual; Informatics; Isotopes; K-562; K562 Cells; Lead; Libraries; Link; MALDI-TOF Mass Spectrometry; Measurement; Measures; Methodology; Methods; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Molecular Bank; Noise; Numbers; Oncogenic; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Preparation; Protein Kinase; Protein Tyrosine Kinase; Proteins; Proto-Oncogene Protein c-kit; Rate; Reagent; Receptor Protein-Tyrosine Kinases; Reporting; Reproducibility; Research Personnel; Resolution; SU11248; Sampling; Scanning; Screening procedure; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Signaling Protein; Solid; Specificity; Speed; Spottings; Statistically Significant; Surface; Testing; Time; Training; Tyrosine Phosphorylation; Validation; Work; cell growth; copolymer; falls; high throughput screening; in vivo; inhibitor/antagonist; instrumentation; leukemia; lung small cell carcinoma; miniaturize; novel; novel strategies; programs; protein activation; receptor; research study; response; small molecule; small molecule libraries; tool

DESCRIPTION (provided by applicant): In response to RFA-RM-04-020, "Molecular Libraries Screening Instrumentation", we propose to develop a highly integrated hardware and software platform that performs highly multiplexed high throughput assays for signaling protein activation in extracts from cells treated with individual compounds. Current high throughput screening methods offer powerful tools to discover novel modulators of cell signaling in large libraries of small molecules by detecting the chemical modification of substrates by kinases, phosphatase, proteases and other signaling enzymes. This approach has proven its value in discovering inhibitors of oncogenic tyrosine kinases, leading to clinically important drugs such as Imatinib to target Bcr-Abl in CML and c-kit in GIST, Gefitinib to target EGFR in small cell lung cancer and a number of investigational kinase antagonists such as SU11248 to target Flt3 in AML. However, the state-of-the-art in molecular library screening falls short in identifying molecules that may be agonists or antagonists of specific signaling pathways but that are not necessarily targeted at a particular signaling protein. In prior work, we have developed sensitive assays using immunologic and MALDI-TOF detection of the phosphorylation of immobilized peptides and proteins that accurately report Bcr-Abl activity and inhibition in whole cell extracts from Ph+ leukemia cell lines. Here, we will extend this work and use our established copolymerization methodology to immobilize multiple tyrosine kinase substrate and isotope-coded control peptides in an acrylamide copolymer via photo-cleavable linkers. Peptides that display high specificity for individual tyrosine kinases or specific classes will be selected and validated in this format. Cell extracts will be applied to these surfaces in an array format where each spot corresponds to cells treated with a single library compound. After incubation, the surface will be washed, and the peptides released by UV photo-cleavage. MALDI-TOF analysis of each spot will lead to a spectrum of test and control peptides associated with each compound. These spectra will be normalized and compared with reference spectra to detect statistically significant changes in one or more signaling pathways. Compounds that appear to activate or inhibit one or more tyrosine kinases present in the cell extracts will be flagged for further analysis.

描述（由申请人提供）：为了响应 RFA-RM-04-020“分子文库筛选仪器”，我们建议开发一个高度集成的硬件和软件平台，该平台可对提取物中的信号蛋白激活进行高度多重高通量测定。用单独的化合物处理的细胞。目前的高通量筛选方法提供了强大的工具，通过检测激酶、磷酸酶、蛋白酶和其他信号酶对底物的化学修饰，在大型小分子文库中发现细胞信号传导的新型调节剂。这种方法已证明其在发现致癌酪氨酸激酶抑制剂方面的价值，从而产生临床上重要的药物，例如针对 CML 中的 Bcr-Abl 的伊马替尼和针对 GIST 中的 c-kit、针对小细胞肺癌中的 EGFR 的吉非替尼以及许多研究性药物。激酶拮抗剂如 SU11248 在 AML 中靶向 Flt3。然而，分子库筛选的最新技术在鉴定可能是特定信号传导途径的激动剂或拮抗剂但不一定针对特定信号传导蛋白的分子方面存在不足。 在之前的工作中，我们开发了使用免疫学和 MALDI-TOF 检测固定化肽和蛋白质磷酸化的灵敏测定法，准确报告 Ph+ 白血病细胞系全细胞提取物中的 Bcr-Abl 活性和抑制作用。在这里，我们将扩展这项工作，并使用我们已建立的共聚方法通过光可裂解的连接体将多种酪氨酸激酶底物和同位素编码的控制肽固定在丙烯酰胺共聚物中。对单个酪氨酸激酶或特定类别表现出高特异性的肽将以这种形式进行选择和验证。细胞提取物将以阵列形式应用于这些表面，其中每个点对应于用单一库化合物处理的细胞。孵育后，清洗表面，并通过紫外光裂解释放肽。每个点的 MALDI-TOF 分析将产生与每种化合物相关的测试和对照肽谱。这些光谱将被归一化并与参考光谱进行比较，以检测一个或多个信号传导途径中统计上显着的变化。似乎激活或抑制细胞提取物中存在的一种或多种酪氨酸激酶的化合物将被标记以供进一步分析。