Integral Membrane Proteins and Lipids Ejected from the Membranes of Native Tissues

我们的总体目标是使在天然环境中研究膜蛋白成为可能。我们将使用新的质谱学(MS)方法实现哺乳动物膜蛋白组件从其自然组织环境中完整地喷射出来(而不首先溶解双层)以进行询问。这将使我们能够将膜蛋白的小分子调节与蛋白质的真实修饰状态联系起来，这可能包括寡聚、翻译后修饰(PTM)和蛋白质与自身或其他生物分子的聚集。在这样做的过程中，我们将把膜蛋白组件的MS从依赖于使用洗涤剂提取组件的单个组件的方法转变为能够将这些组件作为整体进行分析的方法。它将包括将完整的组件直接从健康和患病状态下的人类细胞和组织的膜中喷射出来。为什么需要这个项目？尽管已经发展了许多高通量和扩展的基因组、转录和蛋白质组学方法来确定疾病的基础，但目前还不可能确定PTMS和单核苷酸多态(SNPs)是否或如何影响膜蛋白与它们在细胞膜中遇到的一系列脂类、辅因子和其他潜在相互作用因子的相互作用。然而，维持这种相互作用--并确定膜蛋白PTM和SNPs如何影响与膜上其他生物分子的相互作用--对于指导治疗选择至关重要。为什么是现在？因为我们刚刚发现了如何通过脂质双层保存信号通路，并拥有新的强大的MS技术，准备发现新的信号通路并提出干预措施。最先进的解决方案方法论：出于在更自然的环境中进行研究的愿望，我们证明了我们可以直接从细菌和线粒体膜中排出蛋白质，而不需要借助化学(洗涤剂)干预。我们通过对脂泡施加短的超声波脉冲(SOLE)并将囊泡碎片直接引入质谱仪来实现这一点。使用这种方法，我们发现了细菌复合体亚基组成的差异(c.f.胶束中过表达的蛋白质)，以及意想不到的脂质和辅因子结合。在我们最新的SOLE应用中，我们捕获了牛视紫质通过天然膜片段向其下游效应器发出的信号。随着MS的新发展，我们已经开始将小分子与膜蛋白连接起来，我们已经积累了几项关键的技术进步，现在准备实施进一步的MS突破。在我们第一次发表解决方案的时候，我们从牛线粒体中喷射出复合体，我们无法对喷射出的蛋白质进行排序，因此使用辅因子结合和亚单位质量来通知我们的分配。对这种方法的可靠性的质疑促使我们继续与Thermo Fisher合作，进一步开发Tribrid技术。我们现在可以对完整的复合体进行自上而下的测序，这样我们就可以确认它们的身份，并将小分子结合与真实自然环境中蛋白质的PTM状态联系起来。Thermo Fisher的持续开发需要密切合作--首先解决洗涤剂胶束中膜蛋白复合体的喷射问题，然后开发膜蛋白复合体从膜中的喷射。我们已经提交了两项联合专利申请，并获得了我们帮助设计的原型仪器的使用权，该仪器将于2022年夏天安装在我们的实验室中。该仪器配备了红外线激光器，使我们能够同步激光曝光时间和功率，使我们能够微调组件从膜环境中的喷射。

Our overall objective is to make it possible to study membrane proteins in their native context. We will implement the ejection of mammalian membrane protein assemblies intact from their native tissue environments (without first dissolving the bilayer) for interrogation using new mass spectrometry (MS) approaches. This will enable us to connect small-molecule regulation of membrane proteins to the true modified status of the protein, which may include oligomerisation, post-translational modifications (PTMs) and clustering of the protein with itself or other biomolecules. In so doing, we will transition MS of membrane protein assemblies from an approach that relies on using detergent to extract individual components of assemblies to one capable of analysing those components as an ensemble. It will involve directly ejecting intact assemblies from the membranes of human cells and tissues in healthy and diseased states. Why is this programme needed? Despite the development of numerous high-throughput and expansive genomic, transcriptomic, and proteomic methods to identify the underpinnings of disease, it is currently not possible to define whether, or how, PTMs and single-nucleotide polymorphisms (SNPs) impact membrane proteins' interactions with the array of lipids, cofactors and other potential interactors they encounter in cell membranes. Maintaining such interactions - and defining how membrane protein PTMs and SNPs affect interactions with other biomolecules in the membrane - is, however, critical for informing therapeutic choices. Why now? Because we have just discovered how to preserve a signalling pathway across a lipid bilayer and have new powerful MS technology ready to uncover new signalling pathways and propose interventions. State-of-the-art SoLVe methodology: Motivated by the desire to conduct research in ever-more-native environments, we demonstrated that we could eject proteins directly from bacterial and mitochondrial membranes without recourse to chemical (detergent) intervention. We achieved this by applying short Sonication pulses to Lipid Vesicles (SoLVe) and introducing vesicle fragments directly into a mass spectrometer. Using this approach, we uncovered differences in the subunit composition of bacterial complexes (c.f. over-expressed proteins in micelles), and unexpected lipid and cofactor binding. In our most recent application of SoLVe, we captured bovine rhodopsin signalling to its downstream effectors across native membrane fragments. Together with novel MS developments, in which we have begun to link small molecules with membrane proteins, we have amassed several critical technological advances and are now poised to implement further MS breakthroughs. At the time of our first SoLVe publication, in which we ejected complexes from bovine mitochondria, we were unable to sequence the ejected proteins and so used co-factor binding and subunit masses to inform our assignments. Questions raised about the reliability of this approach prompted us to continue collaborating with Thermo Fisher to develop further the Tribrid technology. We can now apply top-down sequencing to intact complexes such that we can confirm their identity and link small molecule binding to the PTM status of proteins in true native environments. Continued development with Thermo Fisher has involved close collaboration - first to troubleshoot the ejection of membrane protein complexes from detergent micelles and then to develop ejection from membranes. We have filed two joint patent applications and secured access to the prototype instrument that we helped design, to be installed in our laboratory in the summer of 2022. Equipped with an infra-red laser, allowing us to synchronise laser exposure time and power, the instrument enables us to fine-tune the ejection of assemblies from membrane environments.