Understanding organization of membrane proteins and lipids through lipid vesicle native mass spectrometry

摘要 在拥挤的膜环境中，膜蛋白与其他可溶性和膜相关 蛋白质和脂类形成大量动态和瞬时的蛋白质复合体，这些复合体反过来又控制着细胞 生理学。越来越多的证据表明，独立的膜蛋白-脂质相互作用以及 宿主膜的整体生物物理性质通常调节这些组装。因此，要了解如何 特定膜蛋白之间的联系有助于细胞对外部刺激的反应，我们需要研究 从脂类双层环境中直接组装相应蛋白质的寡聚体。这将把我们带到 研究膜蛋白-脂质相互作用的主要挑战。现有的研究此类问题的工具 相互作用缺乏这种直接从双层环境进行分子分析的关键能力。 为了应对这一挑战，该项目的总体目标是开发一个新颖的实验平台， 能够直接从体外脂质双层中分析MP复合体，这种双层可以针对目标细胞定制 薄膜。为此，我们将结合脂泡技术和天然质谱学(NMS)。在AIM 1、选取10种不同的标准寡聚膜蛋白，我们将开发一种实验方法 这使我们能够直接从一系列模拟不同类型的脂泡中确定它们的低聚状态 生理膜。我们将根据已知的寡聚体质量来验证我们的结果并将其作为基准 这些蛋白质中的每一种。这将建立我们的平台的适用性，以检测广泛的薄膜 来自各种脂类双层环境的蛋白质。在目标2中，我们将开发一种实验性策略， 使我们能够直接确定特定结合的脂类结合以及它们结合的位置。为此，我们将 将NMS和基于脂类组学的高效液相色谱-MS/MS分析相结合，以确定脂类的身份。同时，在 与Thermo Fisher Science合作，我们将把ECD碎裂与脂泡NMS平台结合起来 确定脂质结合部位。成功完成后，这两个目标将共同提供 阿森纳的新技术，直接研究膜蛋白和脂类的寡聚组织 一种生理上相关的脂双层。在目标3中，我们将把这一点应用于复杂的生物系统，以解决 神经生物学中的悬而未决的问题；神经递质填充的突触小泡如何达到其超快的速度 核聚变。为此，我们将特别针对突触素的作用，突触囊泡膜蛋白 它与各种神经疾病有关。提出的实验可以带来关键的 了解神经元信号转导和相关疾病特异性的机械师和结构洞察力 减损。从长远来看，膜蛋白之间的联系受损与几个因素有关 从神经退化到癌症的各种病理生理状况。我们有信心，拟议的 平台将在研究广泛的生物过程和相关疾病方面发挥变革性的作用 各州。

Abstract In the crowded milieu of the membrane, membrane proteins, with other soluble and membrane-associated proteins, and lipids form a large number of dynamic and transient protein complexes that in turn govern cellular physiology. There is mounting evidence that both independent membrane protein-lipid interactions, as well as bulk biophysical properties of the host membrane often regulate these assemblies. Hence, to understand how associations between specific membrane proteins help a cell responds to an external stimulus, we need to study the oligomeric assemblies of the respective proteins directly from the lipid bilayer environment. This brings us to the primary challenge of studying membrane protein-lipid interactions. The existing tools to study such interactions lack this critical ability to perform molecular analysis directly from the bilayer environment. Addressing this challenge, the overarching goal of this project is to develop a novel experimental platform that enables analysis of MP complexes directly from in vitro lipid bilayers, which can be customized to a target cellular membrane. To this end, we will combine lipid vesicle technologies with native mass spectrometry (nMS). In Aim 1, taking a set of ten different standard oligomeric membrane proteins we will develop an experimental method that enables us to determine their oligomeric states directly from a range of lipid vesicles mimicking different physiological membranes. We will validate and benchmark our results against the known oligomeric masses of each of these proteins. This will establish the applicability of our platform to detect a wide range of membrane proteins from a variety of lipid bilayer environments. In Aim 2, we will develop an experimental strategy that enables us to directly determine the specifically bound lipid binds and where do they bind. To this end, we will combine nMS with HPLC MS/MS analysis based lipidomics to determine the identity of the lipds. In parallel, in collaboration with Thermo Fisher Scientific, we will combine ECD fragmentation with lipid vesicle nMS platform to determine the site of lipid binding. Together, upon successful completion, these two Aims will provide an arsenal of new technologies to study the oligomeric organization of membrane proteins and lipids directly from a physiologically relevant lipid bilayer. In Aim 3, we will apply this to a complex biological system to address an outstanding question in neurobiology; how neurotransmitter filled synaptic vesicles attain their ultrafast speed of fusion. To this end, we will specifically target the role of synaptophysin, a synaptic vesicle membrane protein which has been linked to various neurological disorders. The experiment proposed can bring out critical mechanist and structural insight to understand neuronal signal transduction and related disease-specific impairments. In the long run, impairment of associations between membrane proteins has been linked to several pathophysiological conditions ranging from neurodegeneration to cancer. We are confident that the proposed platform will have a transformative role in studying a wide range of biological processes and associated disease states.