标题： 使用天然质谱法量化蛋白质同源二聚体亲和力 显示英文标题

标题： Quantification of protein homodimer affinity using native mass spectrometry

摘要： 生物过程依赖于(生物大)分子之间的精细调节的同源和异源相互作用。 相互作用的强度，通常由解离常数(KD)表示，在基础研究中起着至关重要的作用，并且在药物和农用化学品的开发过程中必须进行监测。 一种理想的KD测定方法应适用于各种分析物，具有广泛的亲和力范围，能容忍复杂的基质组成，不需要标记，并且同时提供结合伙伴的结构完整性的信息。 天然质谱符合这些标准，但通常在同源寡聚复合物方面存在困难，因为质量信号重叠。 为了解决这个问题，我们通过样品稀释和共价交联分别分析每种寡聚物种的电荷状态分布，从而解析单体/二聚体对重叠MS峰的贡献。 按照这种方法，我们展示了定量激光诱导液滴离子解吸质谱(qLILBID-MS)能够准确捕捉牛血清白蛋白和来自人类病原性布氏锥虫的氧化还原酶Tryparedoxin的化学诱导二聚体与各种分子胶和同源二聚体亲和力。 方便的是，qLILBID-MS所需的样品量仅为其他方法如等温滴定量热法所用样品的一小部分，并能够获得以前无法获取的高微摩尔范围内的蛋白质同源二聚体KD，这使我们能够通过关键二聚体界面接触的突变来监测同源二聚体亲和力的逐渐降低。 总体而言，qLILBID-MS是一种灵敏、稳健、快速、可扩展且成本效益高的替代方法，用于量化蛋白质/蛋白质相互作用，可以加速当代药物发现工作流程，例如高效筛选邻近诱导分子，如蛋白降解靶向嵌合体和分子胶。 显示英文摘要

摘要： Biological processes rely on finely tuned homo- and heteromeric interactions between (biomacro)molecules. The strength of an interaction, typically given by the dissociation constant (KD), plays a crucial role in basic research and must be monitored throughout the development of drugs and agrochemicals. An ideal method for KD determination is applicable to various analytes with a large range of affinities, tolerates complex matrix compositions, does not require labeling, and simultaneously provides information on the structural integrity of the binding partners. Native mass spectrometry meets these criteria but typically struggles with homooligomeric complexes due to overlapping mass signals. To overcome this, we resolve monomer/dimer contributions to overlapping MS-peaks by separately analyzing the charge state distribution of each oligomeric species via sample dilution and covalent crosslinking. Following this approach, we show that quantitative Laser-Induced Liquid Bead Ion Desorption mass spectrometry (qLILBID-MS) accurately captures the affinities of Bovine Serum Albumin and chemically induced dimers of Tryparedoxin, an oxidoreductase from human pathogenic Trypanosoma brucei parasites, with various molecular glues and homodimer affinities. Conveniently, qLILBID-MS requires a fraction of sample used by other methods such as isothermal titration calorimetry and yields previously inaccessible protein homodimer KDs in the high micromolar range, which allowed us to monitor the gradual decrease in homodimer affinity via mutation of crucial dimer interface contacts. Overall, qLILBID-MS is a sensitive, robust, fast, scalable, and cost-effective alternative to quantify protein/protein interactions that can accelerate contemporary drug discovery workflows, e.g. the efficient screening for proximity inducing molecules like proteolysis targeting chimera and molecular glues.