Native ambient mass spectrometry for membrane proteins

蛋白质是大的生物分子，执行生命所需的所有功能。蛋白质的无数作用和用途不仅仅取决于它们的化学结构，还取决于它们折叠成的形状。因此，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，它可以提供化学和整体三维结构的信息。质谱法就是这样一种工具，它为蛋白质的运作机制提供了惊人的见解。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纠缠在一起，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通往细胞内部的通道，因此占到高血压、心脏病、中风和癌症等疾病药物靶点的70%左右。膜蛋白也可以被病毒劫持，促进它们进入细胞，并再次为治疗方法的发展提供焦点。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形状的挑战，膜蛋白的结构信息落后于更容易获得的蛋白质。我们开发了原生环境质谱（NAMS），这是一种直接从组织中分析蛋白质及其相互作用伙伴的分子工具。NAMS既提供结构信息，又使其空间分布可视化。迄今为止，NAMS主要应用于可接近的可溶性蛋白质。然而，初步数据表明，该方法也适用于膜蛋白的成像和分析。本提案的目的是开发专门针对膜蛋白的NAMS。我们将设计、构建和验证NAMS的来源，使试剂的靶向递送能够促进从组织中提取膜蛋白。将研究两种提取方法：1)使用溶解洗涤剂和2)在“苯乙烯马来酸脂质颗粒”或small中捕获膜蛋白和一些周围膜分子。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.