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）：为响应RFA-RM-04-020“分子库筛选仪器”，我们提出开发一种高度集成的硬件和软件平台，该平台可进行高度多重高通量测定，用于在用单个化合物处理的细胞提取物中发出蛋白质活化信号。目前的高通量筛选方法提供了强有力的工具，通过检测激酶、磷酸酶、蛋白酶和其他信号传导酶对底物的化学修饰，在大的小分子文库中发现细胞信号传导的新调节剂。这种方法已经证明了其在发现致癌酪氨酸激酶抑制剂方面的价值，导致临床上重要的药物如伊马替尼靶向CML中的Bcr-Abl和GIST中的c-kit，吉非替尼靶向小细胞肺癌中的EGFR，以及许多研究性激酶拮抗剂如SU 11248靶向AML中的Flt 3。然而，分子文库筛选的最新技术水平福尔斯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靶向特定信号传导蛋白的分子方面不足。 在以前的工作中，我们已经开发了灵敏的检测方法，使用免疫学和MALDI-TOF检测的磷酸化的固定化肽和蛋白质，准确地报告Bcr-Abl活性和抑制在全细胞提取物的Ph+白血病细胞系。在这里，我们将扩展这项工作，并使用我们建立的共聚方法，通过光可裂解的连接器，在丙烯酰胺共聚物中的多个酪氨酸激酶底物和同位素编码的控制肽。将选择对单个酪氨酸激酶或特定类别显示高特异性的肽，并以这种形式进行验证。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文库化合物处理的细胞。孵育后，洗涤表面，并通过UV光裂解释放肽。每个斑点的MALDI-TOF分析将产生与每个化合物相关的测试和对照肽的光谱。将这些光谱归一化并与参考光谱进行比较，以检测一种或多种信号传导途径中的统计学显著变化。将标记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用于进一步分析。