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、分辨率和速度现在为发展提供了机会。 能够解决膜的重要结构和动力学问题的自下而上的足迹方法 蛋白质。这种自下而上的方法从根本上来说是一种比自上而下的质量更强大的选择 光谱(MS)研究主要限于细菌膜蛋白。我们关注的是人类 蛋白质，因为它们参与了几乎所有的生理过程，占药物总量的60%以上 目标。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，大约有100个这样的蛋白质的结构是已知的，留下了一个很大的空白，需要MS来填补。我们的 长期目标是开发全面的足迹MS方法，提供独特的结构方法 以及活细胞和体外脂双层中膜蛋白的动态变化。我们的目标是合成 提供高足迹覆盖率以揭示配体相互作用和动态的新化学探针 代表最大膜转运蛋白超家族的模型蛋白--铁蛋白的转运运动 以及维持人类体内的铁平衡。我们的假设是：(1)补充化学可以最大化 覆盖了足迹，从而提高了空间分辨率。此外，调谐物理 标记试剂的性质将允许进入膜蛋白的疏水性区域。(2)照片- 激活的快速足迹可以揭示发生在毫秒内的动态转运体运动，这是 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，这是一种结晶学难以捉摸的结构 或者是冷冻-EM。使用这些传统的方法需要纯化的蛋白质，但大多数膜蛋白是 不够稳定，经不起苛刻的提纯。活细胞足迹完全避免了这个庞然大物 困难重重。我们的假设建立在实验室产生的大量初步数据的基础上。具体来说，我们 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MS足迹可以揭示人膜的配体结合作用的原理 蛋白质存在于脂双层中，并可以报告它们在活细胞中的天然结构状态和运动。为了实现我们的目标 为了实现目标，我们将追求三个具体目标：(1)开发新的化学探测器，以提供高足迹覆盖率 (2)在脂膜体系中应用新的探针研究配体相互作用。 和毫秒运动；以及(3)证明了新的探针与活细胞足迹的兼容性 通过检测细胞运动和铁蛋白的配基相互作用。我们的创新足迹与 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、广泛的足迹 膜。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.