Mass Spectrometry and Enzyme Processes

描述（由申请人提供）：本项目的长期目标是 开发允许分析各种酶的方法， 使用质谱法进行处理。本报告建议的主要领域包括： 1)固定低浓度的不同种类的酶， 效率和保留酶活性，用于筛选目的 大量的抑制剂库（配体）使用我们新开发的IEMS 法 2)确定抑制率至少为40%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， 识别合成副产物， 3)根据需要计算固定化和可溶性的Km、Vmax和kcat 酶使用我们的MS方法。 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。 采用离子阱或FTICR-MS的IEMS方法，无需 层析FTICR的高分辨率和高质量精度将 允许在IEMS测定中分离同量异位素。MS的多个阶段将 提供了两种所需合成产物的结构鉴定， 组合合成和未预料到的合成副产物。是 另外提出，动力学参数可以使用ESI离子计算 阱质谱法，从而避免了需要发色团， 传统的分光光度技术。已经建立了一个合作 与卡罗琳·贝尔托齐教授合作， 磺基转移酶（EST）和糖基磺基转移酶（NoST）将被 除了其他酶系统外，还研究了其他酶系统。

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.