Capturing the organisation of the Mycoplasma pneumoniae proteome with in-cell crosslinking mass spectrometry

细胞具有复杂的内部组织，无法通过离体实验完全捕获。例如，相变和“无膜细胞器”已经通过荧光显微镜和冷冻电子断层扫描显示。最近一篇涉及我们的论文将“蛋白质群落”确定为高于“核心”蛋白质复合物的潜在细胞组织水平，该蛋白质复合物通常在纯化过程中丢失（Kaplanche，O 'Reilly等人2017），并且理想地进行原位研究。关于蛋白质组的这种高级组织，我们知之甚少。目前，我们对蛋白质组如何组织的最全面的理解来自于大规模的蛋白质组学研究，这些研究通过亲和纯化/质谱或共分馏来鉴定蛋白质复合物。重要的是，这些技术不产生所发现的复合物的拓扑信息，因此需要纯化复合物以进行进一步的结构分析，这可能导致相关因子丢失。此外，不溶性或膜相关的复合物通常被遗漏。这些都是细胞内交联质谱（CLMS）可以解决的问题。在这个建议中，我们将分析简单的模型病原体肺炎支原体的蛋白质组的拓扑结构，并发现多远的生物过程组织独立的细胞器。特别是，我们将调查的expressosome作为一个先前确定的“高阶”复杂的M。pneumoniae，其结构信息知之甚少。免疫沉淀实验表明，M.肺炎。然而，没有在原位结构调查已经进行，所以任何相关的因素，这种相互作用是未知的。在细胞CLMS结合综合结构建模技术将建立一个组织模型的M。pneumoniae蛋白质组原位研究分枝肺炎杆菌蛋白质组在这里作为模式生物体特别有用，原因有二：首先，它足够简单，允许通过CLMS进行全面分析;其次，已经进行了许多以前的系统范围的研究，因此是用于验证的有价值的资源。我们的初步工作已经表明，我们可以检测到大量以前未知的相互作用，并可以通过与EMBL的Julia Mahamid小组合作的细胞内冷冻电子断层扫描的强大组合，进一步在结构上验证选定的相互作用。这项研究中收集的系统级结构见解，虽然对细胞生物学家来说很有趣，也将为对抗这种病原体的新疗法指明潜在的靶点。

Cells have complex internal organization which cannot be fully captured via ex vivo experiments. For example, phase transitions and “membrane-less organelles” have been shown by fluorescence microscopy and cryo-electron tomography. A recent paper involving us identified “protein communities” as a potential level of cellular organisation above that of “core” protein complexes that is often lost during purification (Kastritis, O’Reilly et al. 2017) and is ideally studied in situ. Very little is known about this higher-order organisation of the proteome. Currently our most comprehensive understanding for how the proteome is organised has come from large-scale proteomics studies that identified protein complexes by affinity-purification/mass spectrometry or co-fractionation. Importantly these techniques do not produce topological information for the complexes discovered and so complexes have needed to be purified for further structural analysis, potentially causing associated factors to be lost. Additionally, insoluble or membrane associated complexes are usually missed. These are issues that in-cell crosslinking mass spectrometry (CLMS) can address. In this proposal, we will analyze the topology of the proteome of the simple model pathogenic organism Mycoplasma pneumoniae and discover how far biological processes are organised independent of organelles. In particular, we will investigate the expressosome as a previously identified ‘higher-order’ complex in M. pneumoniae for which little structural information is known. Immunoprecipitation experiments have shown that there is direct coupling of transcription and translation in M. pneumoniae. However no in situ structural investigation has been conducted so any associated factors necessary for this interaction are unknown.In-cell CLMS combined with integrative structural modelling techniques will build an organisational model of the M. pneumoniae proteome in situ. The M. pneumoniae proteome is particularly useful here as a model organism for two reasons: firstly, it is simple enough to allow for comprehensive analysis by CLMS; secondly, many previous system-wide studies have been performed and so are a valuable resource for validation. Our preliminary work has already shown that we can detect a large number of previously unknown interactions and can further structurally validate selected interactions through the powerful combination with in-cell cryo-electron tomography in collaboration with the group of Julia Mahamid at EMBL.The systems-level structural insights gathered in this study, while interesting in general for cell biologists, will also indicate potential targets for novel therapies against this pathogen.