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

我们的总体目标是使研究膜蛋白的天然环境成为可能。我们将使用新的质谱（MS）方法从其原生组织环境中完整地喷射哺乳动物膜蛋白组装体（不首先溶解双分子层）进行询问。这将使我们能够将膜蛋白的小分子调控与蛋白质的真正修饰状态联系起来，其中可能包括寡聚化，翻译后修饰（PTMs）以及蛋白质与自身或其他生物分子的聚类。在这样做的过程中，我们将膜蛋白组件的质谱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一个整体进行分析的方法。它将涉及直接从健康和患病状态的人类细胞和组织的膜上射出完整的组件。为什么需要这个项目？尽管发展了许多高通量和广泛的基因组学、转录组学和蛋白质组学方法来识别疾病的基础，但目前还不可能确定ptm和单核苷酸多态性（snp）是否或如何影响膜蛋白与一系列脂质、辅助因子和其他潜在相互作用物在细胞膜上的相互作用。然而，维持这种相互作用，并确定膜蛋白PTMs和snp如何影响与膜中其他生物分子的相互作用，对于告知治疗选择至关重要。为什么是现在?因为我们刚刚发现了如何保存跨脂质双分子层的信号通路，并且有新的强大的MS技术准备发现新的信号通路并提出干预措施。最先进的解决方法：由于希望在越来越原生的环境中进行研究，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，而无需求助于化学（洗涤剂）干预。我们通过对脂质囊泡（SoLVe）施加短超声脉冲并将囊泡片段直接引入质谱仪来实现这一目标。使用这种方法，我们发现了细菌复合物亚基组成的差异（如胶束中过度表达的蛋白质），以及意想不到的脂质和辅因子结合。在我们最近的SoLVe应用中，我们捕获了牛视紫红质通过天然膜片段向其下游效应器传递信号。随着新的质谱发展，我们已经开始将小分子与膜蛋白联系起来，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质谱突破。在我们的第一篇SoLVe论文中，我们从牛线粒体中喷射复合物，我们无法对喷射的蛋白质进行测序，因此使用辅因子结合和亚基质量来通知我们的分配。人们对这种方法的可靠性提出了质疑，促使我们继续与赛默飞世尔合作，进一步开发Tribrid技术。我们现在可以对完整的复合物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正天然环境中蛋白质的PTM状态联系起来。与赛默飞世尔的持续开发涉及密切合作-首先解决洗涤剂胶束中膜蛋白复合物的喷射问题，然后开发膜喷射。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，使我们能够微调膜环境中组件的弹出。

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.