Interrogating the folding and function of membrane proteins by mass spectrometry

膜蛋白是附着在细胞膜上或与细胞膜相联系的蛋白质。膜是细胞不可渗透的表面，许多离子、营养物质、激素、药物和蛋白质通过膜进出细胞，膜蛋白负责跨膜运输和细胞信号传导，后者控制细胞的行为。膜蛋白在医学上具有重要意义。涉及膜蛋白的疾病的例子包括：癌症，可能涉及膜蛋白的过度产生和细胞信号传导途径的错误；心脏病，对离子穿过细胞膜的调节可能失效；糖尿病与膜蛋白突变有关；还有抑郁症，细胞中特定分子的水平不平衡。由于膜蛋白与这些疾病的关联，以及它们在细胞表面的便利位置，小分子药物可以接近它们，膜蛋白占所有已知药物靶点的50%。它们也是抗菌药物的重要靶点。尽管膜蛋白占所有蛋白质的约30%，但其特性和作用方式的细节很少，因为它们是众所周知的难以处理和分析的物种，因为它们的疏水性和不溶性。与水溶性蛋白相比，很少有膜蛋白在结构上被表征：在蛋白质数据库中存档的总共65000个蛋白质结构中，只有309个是膜蛋白。我们对许多疾病的理解，以及我们随后治疗这些疾病和提高我们生活质量的能力，都依赖于对这些蛋白质结构的更好了解，它们与离子和小分子相互作用的方式，以及它们的功能模式。我们的目标是通过开发分析方法来了解膜蛋白的功能方式，我们可以监控折叠或功能事件期间发生的结构变化。我们将使用电喷雾电离-质谱法（ESI-MS）和离子迁移率光谱法，这是一种结合技术，可以在一次快速实验中产生复杂混合物中单个蛋白质物种的质量、化学计量、形状和稳定性等信息。它非常适合监测蛋白质改变其3D形状的方式，以及监测蛋白质和其他物种之间的反应，其中可以同时分析起始材料，反应中间体和产物。我们开创了这项技术，以实时提供对水溶性蛋白功能事件的见解，现在希望将其用于膜蛋白。在ESI-MS分析之前，我们将使用化学标记技术在功能事件的不同时间探测膜蛋白的3D结构。这种方法改变了蛋白质，导致我们可以检测到的质量变化。然后，我们将使用ESI-MS分析反应中的单个物种，并准确确定蛋白质的哪些区域暴露并可用于反应（标记区域），以及那些在其3D结构中被屏蔽或与其他物种相互作用的区域。通过在反应过程中的不同时间进行标记和分析，我们将实时生成一系列事件的“快照”。这些快照将提供一个完整的画面，揭示膜蛋白的功能特性。为了进行化学标记和ESI-MS分析，我们将购买激光设备与我们现有的质谱仪耦合，然后开发可应用于广泛膜蛋白的稳健协议。在这项开发工作中，我们选择了两种不同的蛋白质，每一种都是两大类膜蛋白中的一种，即我们有相当丰富的处理经验的全部-螺旋和-桶。

A membrane protein is a protein that is attached to, or associated with, the membrane of a cell. The membrane is the cell's impermeable surface through which many ions, nutrients, hormones, drugs and proteins enter and exit the cell, and membrane proteins are responsible for transportation across the membrane and cell signalling, the latter of which governs the actions of the cell. Membrane proteins are important medically. Examples of maladies in which membrane proteins are implicated include: cancer, which can involve over-production of membrane proteins and errors in cell signalling pathways; heart disease, where the regulation of ions across the cell membrane may be failing; diabetes, which has been linked with membrane protein mutations; and depression, where there is an inbalance of the levels of specific molecules in cells. Due to their association with such diseases, and their convenient location at the cell surface where they are accessible to small molecule drugs, membrane proteins account for >50 % of all known drug targets. They are also important targets for anti-bacterial agents. Despite membrane proteins representing ~30% of all proteins, details of their characteristics and mode of action are scarce as they are notoriously difficult species to handle and analyse because of their hydrophobicity and insolubility. Compared with water-soluble proteins, few membrane proteins have been characterised structurally: only ~309 out of a total of >65,000 protein structures archived in the Protein Data Bank are of membrane proteins. Our understanding of many diseases, and our subsequent ability to treat these diseases and improve our quality of life, is reliant on a better knowledge of the structure of these proteins, the ways in which they interact with ions and small molecules, and their mode of function. Our aim is to understand the way in which membrane proteins function by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. We will use electrospray ionisation-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry, a combined technique which produces information about mass, stoichiometry, shape and stability of individual protein species present within complex mixtures in a single, rapid experiment. It is ideal for monitoring the way a protein changes its 3D shape and for monitoring reactions between proteins and other species, where the starting materials, reaction intermediates and products can be analysed simultaneously. We have pioneered this technique to provide insights into water-soluble protein functioning events in real-time and now wish to exploit its use for membrane proteins. Prior to ESI-MS analysis we will use chemical labelling techniques to probe the 3D structures of membrane proteins at different times during a functioning event. Such methods modify the protein which results in a change of mass which we can detect. We will then use ESI-MS to analyse the individual species within the reaction and identify exactly which regions of the protein are exposed and available to react (the labelled areas), and those regions which are shielded within its 3D structure or by interactions with other species. By labelling and analysing at different times during the reaction we will produce a series of "snapshots" of the event in real-time. Together the snapshots will provide a complete picture revealing insights into the functional properties of membrane proteins. To carry out the chemical labelling and ESI-MS analysis we will purchase laser equipment to couple to our existing mass spectrometers and then develop robust protocols that we can apply to a wide range of membrane proteins. For this development work, we have chosen two different proteins, each an example from one of the two major classes of membrane proteins, the all alpha-helical and beta-barrel, which we have considerable experience of handling.