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

在化学系化学测量和成像（CMI）项目的支持下，杜克大学Michael Fitzgerald教授和他的研究小组将开发和应用新的基于定量质谱的蛋白质组学方法，用于蛋白质-配体结合相互作用的大规模热力学分析。正在开发的特定方案利用氧化速率蛋白质稳定性（SPROX）技术与细胞培养氨基酸稳定同位素标记（SILAC）方法相结合，比较真实生物混合物（例如细胞裂解物）中蛋白质和蛋白质配体复合物的平衡展开/折叠特性。新方案旨在显著扩大蛋白质组范围内SPROX实验中检测的蛋白质数量，将在几个原理验证实验中作为基准，在这些实验中，酵母细胞溶解物中的内源性蛋白质被分析与ATP（一种普遍存在的酶辅因子）、格尔达霉素（一种Hsp90抑制剂）和环孢素a（一种直接结合亲环蛋白a并间接与钙调磷酸酶相互作用的免疫抑制剂）的结合。新方案还将用于真正的药物作用模式研究，涉及马纳桑汀a，这是一种天然产物，在基于细胞的分析中显示出抗癌活性，但作用模式尚不清楚。要创建的策略和方案将很容易地转移到几乎任何蛋白质-配体系统或生物混合物中，而不考虑配体（即小分子，DNA，肽或其他蛋白质）或结合亲和力。该研究为研究生和本科生以及来自北卡罗来纳项目SEED项目的高中生提供了跨学科的研究经验，该项目是一个正在进行的科学充实计划，旨在引导北卡罗来纳高中的弱势、代表性不足的少数民族学生进入化学和化学相关的职业。

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.