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的 蛋白质和脂质形成大量动态和瞬时蛋白质复合物， physiology.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，为了了解 特定膜蛋白之间的联系有助于细胞对外部刺激做出反应，我们需要研究 直接来自脂质双层环境的各个蛋白质的寡聚组装体。这把我们带到 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种新颖的实验平台， 能够直接从体外脂质双层中分析MP复合物，其可以定制为靶细胞 膜的为此，我们将联合收割机脂质囊泡技术与天然质谱（nMS）相结合。在Aim中 1，取一组十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脂质囊泡的脂质囊泡中确定它们的低聚状态。 生理膜我们将验证和基准我们的结果对已知的低聚质量的 每一种蛋白质。这将建立我们的平台的适用性，以检测广泛的膜 来自各种脂质双层环境的蛋白质。在目标2中，我们将开发一种实验策略， 使我们能够直接确定特异性结合的脂质结合以及它们在哪里结合。为此我们将 基于脂质组学的联合收割机nMS与HPLC MS/MS分析相结合，以确定脂质的同一性。同时，在 与Thermo Fisher Scientific合作，我们将联合收割机ECD片段化与脂质囊泡nMS平台相结合 以确定脂质结合的位点。成功完成后，这两个目标将共同提供一个 一系列新技术可以直接从膜蛋白和脂质中研究膜蛋白和脂质的寡聚体组织， 生理学相关的脂质双层。在目标3中，我们将把它应用到一个复杂的生物系统中， 神经生物学中的一个突出问题;充满神经递质的突触囊泡如何达到超快的速度， 核聚变为此，我们将专门针对突触囊泡膜蛋白突触素的作用 它与各种神经系统疾病有关。提出的实验可以带来关键的 机制和结构的洞察力，了解神经元信号转导和相关的疾病特异性 损伤从长远来看，膜蛋白之间的联系受损与几个 从神经变性到癌症的病理生理状况。我们相信，拟议的 该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作用 states.

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.