UVPD Mass Spectrometry of Protein Complexes

抽象的。蛋白质相互作用调节细胞通讯、信号传递和众多其他细胞 功能。对这些相互作用的认识激发了药物发现领域的许多活动 在设计选择性结合和破坏蛋白质靶标功能的抑制剂的背景下。这个 随着耐药菌株的威胁越来越大，药物发现过程面临新的挑战 病原体和寻找更具体的治疗方法来抗击癌症。突破性进展 在对细胞生物化学的理解方面的进步以及在 有助于描述蛋白质相互作用和复合体结构的生物物理方法。 这项建议的重点是发展紫外线光解(UVPD)与 天然喷雾质谱仪作为蛋白质复合体分析的创新策略。这个 蛋白质-配体和蛋白质-蛋白质复合体的鉴定和表征提供了机会 在分子水平上提供对蛋白质功能的洞察并加速药物发现。目标 这项建议的内容包括： 目的1：建立用于检测蛋白质-配体复合体的UVPD。我们将发展壮大 原生喷雾/UVPD-MS策略创建蛋白质-配体复合体的构象图 通过不同的绑定模式。配体的类别将包括底物、辅因子和抑制剂。 目的2：将分离方法与UVPD-MS结合用于蛋白质的高通量分析 复合体。为了扩展UVPD-MS策略的多功能性，我们将对其进行调整以实现更高的吞吐量 与尺寸排除层析(SEC)集成的工作流程。前端SEC将制作样品 介绍可重复性和自动化，从而使其更适合定量应用。 目的3.将UVPD-MS扩展到蛋白质-蛋白质复合体。许多蛋白质存在，并作为一部分发挥作用 因此激发了我们对扩展UVPD-MS方法进行表征的兴趣 蛋白质-蛋白质和蛋白质-蛋白质-配基复合体。 目的4：UVPD-MS方法在解决关键生物学问题中的应用。中开发的方法 AIMS 1-3将通过与以下机构的合作来解决一些高影响的生物问题 分子生物学、生物化学和药物化学研究小组。这些协作 问题包括三个主题：作为药物的蛋白质抑制剂的发展，揭示其影响 蛋白质结构上的点突变，并破译蛋白质-蛋白质相互作用如何调节活性 和功能。

Abstract. Protein interactions mediate cellular communication, signaling, and numerous other cell functions. Recognition of these interactions has inspired much of the activity in the field of drug discovery in the context of designing inhibitors to selectively bind and disrupt the functions of protein targets. The drug discovery process faces new challenges with the growing threat of drug-resistant strains of pathogens and the search for more specific therapeutics to fight cancer. Breakthroughs have been propelled by progress in the understanding of cell biochemistry as well as technological advances in biophysical methods that facilitate the characterization of protein interactions and structures of complexes. This proposal focuses on the development of ultraviolet photodissociation (UVPD) in conjunction with native-spray mass spectrometry as an innovative strategy for analysis of protein complexes. The identification and characterization of protein-ligand and protein-protein complexes offers the opportunity to provide insight into protein function at a molecular level and accelerate drug discovery. The objectives of this proposal include: Aim 1: Development of UVPD for examining protein-ligand complexes. We will develop robust native-spray/UVPD-MS strategies to create conformational maps of protein-ligand complexes governed by different binding modes. The classes of ligands will include substrates, cofactors, and inhibitors. Aim 2: Integrating separation methods with UVPD-MS for higher throughput analysis of protein complexes. To expand the versatility of the UVPD-MS strategy, we will adapt it for a higher throughput workflow via integration with size exclusion chromatography (SEC). Front-end SEC will make sample introduction reproducible and automated, thus making it better suited for quantitative applications. Aim 3. Extending UVPD-MS for protein-protein complexes. Many proteins exist and function as part of multimeric complexes, thus motivating our interest in extending UVPD-MS methods for characterization of protein-protein and protein-protein-ligand complexes. Aim 4: Application of UVPD-MS methods for key biological problems. The methods developed in Aims 1 – 3 will be utilized to attack a number of high impact biological problems via collaborations with research groups in molecular biology, biochemistry, and medicinal chemistry. These collaborative problems encompass three themes: development of protein inhibitors as drugs, unravelling the influence of point mutations on protein structure, and deciphering how protein-protein interactions modulate activity and function.