LC-MS-MS

基于van Breemen博士实验室发明的脉冲超滤质谱法，天然产物提取物将被快速筛选，以发现与重要目标分子结合的特定化合物。这些高通量筛选分析将提高整个程序的生产力。将利用现有的环氧化酶-2和雌激素受体- α和- β配体的超滤筛选方法，以及将开发的用于筛选雄激素受体和RXR等受体配体的新方法。在超滤质谱筛选过程中获得的命中命中将根据分子量，元素组成（使用精确的质量测量确定），串联质谱（MS-MS）碎片模式进行鉴定，并与UIC合作研究计划中的标准进行比较

Based on the method of pulsed ultrafiltration mass spectrometry, which was invented in the laboratory of Dr. van Breemen, natural product extracts will be screened rapidly for the discovery of specific compounds that bind to important target molecules. These high throughput screening assays will enhance the productivity of the entire program. Existing ultrafiltration screening assays for ligands to cyclooxygenase-2 and the estrogen receptors-alpha and -beta will be utilized as well as new assays that will be developed to screen for ligands to receptors such as the androgen receptor and RXR. Hits obtained during ultrafiltration mass spectrometric screening will be identified based on molecular weight, elemental composition (determined using exact mass measurement), tandem mass spectrometric (MS-MS) fragmentation patterns, comparison with standards available at the UIC Program for Collaborative Research in the Pharmaceutical Sciences, dereplication using the UIC NAPRALERT database of natural products and proton NMR and 13C-NMR analysis of HPLC-purified material. Since this screening approach will identify lead compounds based on binding to a specific target protein, these hits will be assayed in Core B to confirm their biological activity. Next, the Phase I and Phase II metabolism of the lead compounds from all parts of the program will be assessed in vitro by incubation with human hepatocytes and human liver microsomes followed by analysis using LC-MS and LC-MS-MS. The in vivo metabolism of cancer chemoprevention agents will be evaluated by LC-MS and LC-MS-MS analysis of blood and urine samples from animal studies. In addition, pharmacokinetic studies of these compounds will be carried out in animals. In another set of preclinical drug development studies, the human intestinal permeability of each lead compound will be evaluated using the Caco-2 cell monolayer model. Finally, LC-MS and LC-MS-MS will be used to confirm the molecular weights, elemental compositions, fragmentation patterns, and purity of compounds. In summary, this project will interact with all of the other Projects within this program by screening natural product extracts for the rapid identification of ligands for specific target proteins, by providing preclinical evaluation of hepatic metabolism, pharmacokinetics and intestinal permeability of lead compounds, and by providing mass spectrometric characterization of isolated and synthetic compounds.