Fast hybrid mass spectrometer for complexome analyses

申请的timsTOF HT质谱仪将取代迄今为止申请人使用的LTQ Orbitrap XL质谱仪，该质谱仪将因经济和技术原因而关闭。生理学研究所II研究快速信号转导的分子基础在质膜和跨质膜，特别是在哺乳动物大脑的神经元。这种信号转导的空间和时间精度背后的中心组织原理是将蛋白质组装成大分子复合物和网络。为了揭示它们的分子外观和组成，该研究所在过去二十年中开发了几种（功能）蛋白质组学方法，并成功地将它们应用于一系列离子通道、膜受体和转运体。实验工作流程包括(i)从天然组织中分离膜组分，（ii）目标蛋白的亲和纯化（多表位AP-MS，“meAP-MS”），（iii）高分辨率分离蛋白质组分和膜亚分离（复杂体分析或细胞器蛋白质组学），以及（iv）高分辨率纳米高效液相色谱耦合质谱法用于鉴定和定量蛋白质，包括其翻译后修饰，如磷酸化，烷基化或脂质修饰。因此，他们的分析需要在质量分辨率（质量/电荷），灵敏度，动态范围和扫描/周期速度方面的高性能质谱分析，特别是高分辨率复杂体分析（通过“冷冻切片蓝色原生质谱”，csBN-MS技术，最近在研究所开发），这需要扩展测量系列（每个生物样品200-400次测量）具有高分析覆盖率。后者将在所要求的仪器上运行，由于其非常高的速度（与目前可用的仪器直接比较，其性能仍然优越）和扩展动态范围的定量（即在单个测量中获得覆盖4-5个数量级的大量蛋白质/肽的定量数据），非常适合。这种高分辨率的复合物分析首次使人们能够全面了解不同生物系统中蛋白质复合物的分子组织和动力学。因此，获得时间/时间/时间对申请人小组未来的工作/项目至关重要。

The requested timsTOF HT mass spectrometer will replace the LTQ Orbitrap XL mass spectrometer that has so far been used by the applicant and will be shut down for economic and technical reasons. The Institute for Physiology II investigates the molecular basis of fast signal transduction at and across the plasma membrane, in particular of neurons in the mammalian brain. A central organization principle behind the exquisite spatial and temporal precision of this signal transduction is the assembly of proteins into macro-molecular complexes and networks. For unraveling their molecular appearance and composition, the institute has developed several approaches of (functional) proteomics over the last two decades and successfully applied them to a series of ion channels, membrane receptors and transporters. The experimental workflows comprise (i) isolation of membrane fractions from native tissues, (ii) affinity-purification of target proteins (multi-epitope AP-MS, "meAP-MS"), (iii) highly resolved separation of protein fractions and membrane sub-fractionations (complexome analysis or organellar proteomics), and (iv) high-resolution nano-HPLC coupled mass spectrometry for identification and quantification of proteins including their post-translational modifications like phosphorylation, alkylation or lipid modifications. Accordingly, their analysis requires high-performance mass spectrometry with respect to mass resolution (mass/charge), sensitivity, dynamic range and scan/cycle speed, in particular for high-resolution complexome analyses (by the ‘cryo-slicing blue native MS‘, csBN-MS technique, recently developed at the institute), which require extended measurement series (200-400 measurements per biological sample) with high analytical coverage. The latter will be run on the requested instrument which - due to its very high speed (>5fold with still superior performance in direct comparison with the presently available instruments) and quantification over an extended dynamic range (i.e. obtaining quantitative data for a larger number of proteins/peptides covering 4-5 orders of magnitude within an individual measurement) - is ideally suited. Such high-resolution complexome analyses enable for the first time a comprehensive view on the molecular organization and dynamics of protein complexes in different biological systems. Acquisition of the timsTOF HT is, therefore, of key importance for the future work/projects of the applicant's group.