Mass Spectrometry and Enzyme Processes

质谱分析和酶处理

• 批准号: 6857144
• 负责人: JULIE Ann LEARY
• 金额: $ 19.55万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 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 2006-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857144
• 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