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.
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