Expanding the Scope of a Chemical Modification and Mass Spectrometry-Based Method For Protein-Ligand Binding Analysis

扩大基于化学修饰和质谱的蛋白质-配体结合分析方法的范围

• 批准号: 1308093
• 负责人: Michael Fitzgerald
• 金额: $ 40.7万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1308093&HistoricalAwards=false
• 关键词: Expanding; Scope; Chemical; Modification; Mass

With support form the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry, Professor Michael Fitzgerald and his research group at Duke University will develop and apply new quantitative mass spectrometry-based proteomics methods for the large-scale thermodynamic analysis of protein-ligand binding interactions. The specific protocols under development utilize the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with a stable isotope labeling with amino acids in cell culture (SILAC) approach to compare the equilibrium unfolding/folding properties of proteins and protein ligand-complexes in real biological mixtures (e.g., cell lysates). The new protocols, which are designed to significantly expand the number of proteins assayed in proteome-wide SPROX experiments, will be benchmarked in several proof-of-principle experiments in which the endogenous proteins in a yeast cell lysate are analyzed for binding to ATP (a ubiquitous enzyme cofactor), to geldanamycin (an Hsp90 inhibitor), and to cyclosporine A (an immunosuppressive agent that directly binds cyclophilin A and indirectly interacts with calcineurin. The new protocols will also be used in a real drug mode-of-action studies involving manassantin A, which is a natural product with demonstrated anti-cancer activity in cell-based assays but an as yet unknown mode-of-action.The strategies and protocols to be created will be readily transferable to almost any protein-ligand system or biological mixture regardless of the ligand (i.e. small molecule, DNA, peptide, or other proteins) or binding affinity. The research provides interdisciplinary research experiences for both graduate and undergraduate students, and as well as high school students from the North Carolina Project SEED program, which is an on-going science enrichment program designed to steer disadvantaged, underrepresented minority students from North Carolina high schools into chemistry and chemistry-related careers.

借助支持形式，化学测量和成像（CMI）在化学划分中，迈克尔·菲茨杰拉德（Michael Fitzgerald）教授及其在杜克大学的研究小组将开发并采用新的基于定量质谱的蛋白质组学方法，以对蛋白质依赖性结合相互作用进行大规模热力学分析。开发中的特定方案利用蛋白质从氧化速率（SPROX）技术与稳定的同位素标记与氨基酸在细胞培养方法（SILAC）方法中结合使用，以比较实际生物学混合物中蛋白质配体的蛋白质和蛋白质配体 - 浓度的平衡/折叠/折叠特性。新方案旨在显着扩大全蛋白质组SPROX实验中测定的蛋白质的数量，将在几个原则实验证明中标记，其中对酵母细胞裂解物中的内源性蛋白质进行分析，以分析与无处不在的enzyme cofactor and spt an hanganammycinnamymycinycin，andssp to atp top top环孢霉素A（一种直接结合环胞活蛋白A并间接与钙调蛋白相互作用的免疫抑制剂。新方案也将用于涉及Manassantin A的真实药物行动模式研究中，该研究是一种自然产品，这是一种自然产品，是一种在基于细胞的模式中证明的抗癌动作，但仍是不知所措的策略。生物混合物不管配体（即小分子，DNA，肽或其他蛋白质）或结合亲和力。和与化学有关的职业。