Mass Spectrometry and Enzyme Processes

描述(申请人提供)：本项目的长期目标是 允许分析各种酶的方法的发展 使用质谱学的过程。本文提出的主要领域包括： 1)固定化不同种类酶的低浓度和高浓度 为了筛选目的而保持酶活性的效率和保持率 使用我们新开发的IEMS的大量抑制剂文库(配体) 方法。 2)确定具有至少40%抑制作用的可能的抑制剂，以及 根据需要应用MS和MS/MS以确保正确的组合合成和 鉴定合成副产品， 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 不需要离子陷阱或FTICR-MS的IEMS方法 层析法。FTICR的高分辨率和高质量精度将 允许在IEMS分析中分离同量异构体。多发性硬化症的多个阶段 提供所需的两种合成产品的结构鉴定 组合合成和意想不到的合成副产品。它是 此外，还提出可以使用ESI离子计算动力学参数 因此，陷阱质谱学不再需要 传统的分光光度技术。已经建立了一项合作 与Carolyn Bertozzi教授一起研究涉及雌激素的项目 磺基转移酶(EST)和糖基转移酶(NOST)将 除了研究其他酶系统外，还进行了研究。

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.