Native ambient mass spectrometry for membrane proteins

蛋白质是一种大的生物分子，可以执行生命所需的所有功能。蛋白质的各种作用和用途不仅取决于它们的化学结构，而且还取决于它们折叠成的形状。因此，对蛋白质的研究需要一种能够在分子水平上进行表征的工具，该工具可以提供有关化学结构和整体三维结构的信息。质谱学就是这样一种工具，它为蛋白质的作用机制提供了惊人的洞察力。这一建议与一类被称为膜蛋白的蛋白质有关。膜蛋白与构成细胞膜的其他分子缠绕在一起，确实需要这些分子来维持它们的形状。它们提供了一条从细胞外部到细胞内部工作的管道，因此在高血压、心脏病、中风和癌症等疾病的药物靶点中占到了约70%。膜蛋白也可以被病毒劫持，促进它们进入细胞，再次为治疗方法的发展提供了焦点。尽管膜蛋白具有潜在的治疗价值，但由于在分析过程中溶解和保持蛋白质的形状存在挑战，有关膜蛋白的结构信息落后于更容易获得的蛋白质。我们开发了自然环境质谱仪(NAMS)，这是一种直接从组织中对蛋白质及其相互作用伙伴进行分子分析的工具。NAMS既能提供结构信息，又能可视化它们的空间分布。到目前为止，NAMS主要应用于可获得的、可溶的蛋白质。然而，初步数据表明，该方法也适用于膜蛋白的成像和分析。这项建议的目的是开发专门针对膜蛋白的NAMS。我们将设计、建造和验证NAMS的来源，使其能够定向递送试剂，以促进从组织中提取膜蛋白。将研究两种提取方法：1)使用增溶洗涤剂；2)将膜蛋白与周围的一些膜分子一起捕获到“苯乙烯-马来酸脂质颗粒”或SMALP中。此外，还将考虑苯乙烯马来酸的替代聚合物。

Proteins are large biomolecules that perform all the functions required for life. The myriad roles and uses of proteins are not simply down to their chemical architecture but also the shapes into which they fold. The study of proteins therefore requires a tool capable of characterization at the molecular level, which provides information on both chemical and overall three-dimensional structure. Mass spectrometry is such a tool, and has provided startling insights into the mechanisms by which proteins operate.This proposal relates to a class of proteins known as membrane proteins. Membrane proteins are entangled with other molecules that make up the cell membrane, indeed need those molecules to maintain their shape. They present a conduit from the outside of a cell to its inner workings, and consequently represent ~ 70% of drug targets for conditions from high blood pressure and heart disease, to stroke and cancer. Membrane proteins can also be hijacked by viruses, facilitating their entry into the cell and again providing a focal point for the development of therapeutics. Despite their potential therapeutic value, structural information on membrane proteins lags behind that available for more accessible proteins because of the challenges of solubilising and maintaining the shape of the protein during analysis. We have developed native ambient mass spectrometry (NAMS), a tool for molecular analysis of proteins and their interacting partners directly from tissue. NAMS both provides structural information and enables their spatial distribution to be visualised. To date, NAMS has been primarily applied to accessible, soluble proteins. Preliminary data, however, suggest that the approach is also suitable for the imaging and analysis of membrane proteins. The aim of this proposal is to develop NAMS specifically for membrane proteins. We will design, build and validate a source for NAMS which enables targeted delivery of reagents to facilitate extraction of membrane proteins from tissue. Two approaches for extraction will be investigated: 1) use of solubilising detergents and 2) capture of membrane proteins together with some of the surrounding membrane molecules in a "styrene maleic acid lipid particle" or SMALP. Alternative polymers to styrene maleic acid will also be considered.