New chemical probes enable Mass Spectrometry-based footprinting of human protein structure in lipid membranes and cells

现代质谱仪的灵敏度、分辨能力和速度现在提供了发展的机会。 自下而上的足迹法能够解决膜的重要结构和动力学问题 proteins.这种自下而上的方法从根本上说是一种比自上而下的质量更强大的替代方法 质谱（MS）研究主要限于细菌膜蛋白。我们专注于人类 蛋白质，因为它们参与几乎所有的生理过程，并代表超过60%的药物 目标的然而，它们是传统高分辨率结构方法最具挑战性的目标。 到目前为止，已知大约100种这些蛋白质的结构，留下了很大的空白供足迹MS填补。我们 长期目标是开发全面的足迹MS方法，提供一种独特的方法来结构 以及活细胞和体外脂质双层中膜蛋白的动力学。我们的目标是合成 新的化学探针，提供高足迹覆盖率，以揭示配体相互作用和动力学 膜转运蛋白的转运运动，膜转运蛋白的最大超家族的模型蛋白 和维持人体内的铁稳态。我们的假设是：（1）互补化学可以最大化 覆盖范围的足迹，从而提高其空间分辨率。此外，调整物理 标记试剂的性质将允许进入膜蛋白的疏水区。(2)照片- 激活的快速足迹可以揭示在毫秒内发生的动态运输机运动， 超出了目前膜结构生物学的范围。(3)生物正交不可逆标记可以 优化以揭示膜蛋白的细胞结构状态，这是晶体学难以理解的结构 或者冷冻电镜这些常规方法的使用需要纯化的蛋白质，但大多数膜蛋白是纯化的。 不足以稳定以承受要求的纯化。活细胞足迹完全避开了这个巨大的 困难我们的假设是建立在我们实验室产生的大量初步数据之上的。我们特别 继续展示我们探索新化学和合成新试剂的能力。我们正在进行的 研究证明了MS足迹法可以揭示人膜的配体结合相互作用的原理 脂质双层中的蛋白质，并且可以报告它们在活细胞中的天然结构状态和运动。完成我们 目标，我们将追求三个具体目标：（1）开发新的化学探针，以提供高足迹覆盖率 （2）在脂质膜系统中实现新的探针以研究配体相互作用 和ferroportin的毫秒运动;和（3）证明新探针与活细胞足迹的兼容性 通过检测细胞运动和膜铁转运蛋白的配体相互作用。我们创新的足迹加上 自下而上的MS蛋白质组学分析将在活细胞和脂质中建立有效的、基础广泛的足迹 膜。所提出的方法的意义将扩大，因为基于MS的足迹可以 被结构蛋白质组学研究人员广泛应用于生物医学上重要的人类膜蛋白。

The sensitivity, resolving power, and speed of modern mass spectrometers now afford the opportunity to develop bottom-up footprinting methods capable of resolving significant structural and dynamics questions of membrane proteins. This bottom-up approach is a fundamentally more powerful alternative to the top-down mass spectrometry (MS) studies that have been mainly limited to bacterial membrane proteins. We focus on human proteins because they participate in almost all physiological processes and represent more than 60% of drug targets. They, however, represent the most challenging targets for traditional high-resolution structural methods. Structures of about 100 of these proteins are known to date, leaving a large gap for footprinting MS to fill. Our long-term goal is to develop comprehensive footprinting MS methods that offer a unique approach to structure and dynamics of membrane proteins in live cells and in vitro lipid bilayers. Our objective here is to synthesize new chemical probes that provide high footprinting coverage to reveal the ligand interaction and dynamic transport motion of ferroportin, a model protein representing the largest superfamily of membrane transporters and maintaining iron homeostasis in humans. Our hypotheses are: (1) Complementary chemistry can maximize the coverage of footprinting and thereby improve its spatial resolution. Furthermore, tuning the physical properties of the labeling reagents will allow access to the hydrophobic region of membrane proteins. (2) Photo- activated fast footprinting can reveal dynamic transporter motions taking place within milliseconds, which is beyond the current scope of membrane structure biology. (3) Bio-orthogonal irreversible labeling can be optimized to reveal the cellular structure state of membrane proteins, a structure that is elusive by crystallography or cryo-EM. Use of these conventional methods requires purified proteins, but most membrane proteins are insufficiently stable to withstand demanding purification. Live-cell footprinting completely avoids this giant difficulty. Our hypotheses are built on extensive preliminary data produced in our laboratories. Specifically, we continue to demonstrate our capability to explore new chemistry and synthesize new reagents. Our ongoing studies prove the principle that MS footprinting can reveal ligand-binding interaction of human membrane proteins in lipid bilayer, and can report on their native structural state and motion in live cells. To accomplish our goals, we will pursue three specific aims: (1) develop new chemical probes to provide high footprinting coverage of membrane proteins; (2) implement the new probes in lipid membrane systems to study the ligand interaction and millisecond motion of ferroportin; and (3) demonstrate the new probes' compatibility with live-cell footprinting by the detection of cellular motions and ligand interactions of ferroportin. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish effective, broad-based footprinting in live cells and lipid membranes. The significance of the proposed approach will expand because MS-based footprinting can be broadly applied by structural proteomics researchers to biomedically important human membrane proteins.