Mass Spectrometry and Enzyme Processes

质谱分析和酶处理

• 批准号: 6625772
• 负责人: JULIE Ann LEARY
• 金额: $ 22.45万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625772
• 关键词: biotechnology; chemical kinetics; combinatorial chemistry; computer data analysis; drug discovery /isolation; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme model; estrogens; high throughput technology; immobilized enzymes; mass spectrometry; model design /development; peptide chemical synthesis; protein purification; statistics /biometry; sulfotransferase; technology /technique development; thermodynamics

DESCRIPTION (provided by applicant): The long-term goal of this project is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Immobilize low concentrations of different classes of enzymes with high efficiency and with retention of enzyme activity for the purpose of screening large numbers of inhibitor libraries (ligands) using our newly developed IEMS method. 2) Identify possible inhibitors showing at least 40 percent inhibition and apply MS and MS/MS as needed to ensure correct combinatorial synthesis and identify synthetic byproducts, 3) Calculate Km, Vmax, and kcat, as needed, of both immobilized and soluble enzymes using our MS method. 4) Determine if inhibition is competitive or non-competitive. 5) Calculate Ki's for inhibitors. It is anticipated that large numbers of compounds can be screened using our IEMS method with either ion trap- or FTICR-MS without the need of chromatography. The high resolution and high mass accuracy of the FTICR will allow for separation of isobars in the IEMS assay. Multiple stages of MS will provide structural identification of both desired synthetic products of the combinatorial synthesis and of unanticipated synthetic byproducts. It is additionally proposed that kinetic parameters can be calculated using ESI-ion trap mass spectrometry thus obviating the need for chromophores as required by traditional spectrophotometric techniques. A collaboration has been established with Professor Carolyn Bertozzi in which projects involving estrogen sulfotransferase (EST) and glycosyl sulfotransferase (NoST) will be investigated in addition to other enzymatic systems.

描述（由申请人提供）：该项目的长期目标是 开发能够分析各种酶的方法 使用质谱分析的过程。本文提出的主要领域包括： 1) 以高浓度固定低浓度的不同类别的酶 效率并保留酶活性以进行筛选 使用我们新开发的 IEMS 获得大量抑制剂库（配体） 方法。 2) 确定显示至少 40% 抑制率的可能抑制剂 根据需要应用 MS 和 MS/MS 以确保正确的组合合成和 识别合成副产品， 3) 根据需要计算固定化和可溶性的 Km、Vmax 和 kcat 使用我们的 MS 方法检测酶。 4) 确定抑制是竞争性的还是非竞争性的。 5) 计算抑制剂的 Ki。 预计可以使用我们的方法筛选大量化合物 IEMS 方法采用离子阱或 FTICR-MS，无需 色谱法。 FTICR 的高分辨率和高质量精度将 允许在 IEMS 测定中分离等压线。 MS 的多个阶段将 提供两种所需合成产物的结构鉴定 组合合成和意想不到的合成副产物。这是 另外提出可以使用 ESI-ion 计算动力学参数 陷阱质谱分析因此不需要发色团 传统的分光光度技术。已建立合作关系 与 Carolyn Bertozzi 教授合作开展涉及雌激素的项目 磺基转移酶 (EST) 和糖基磺基转移酶 (NoST) 除了其他酶系统外还进行了研究。