Fast hybrid mass spectrometer for complexome analyses

用于复杂组分析的快速混合质谱仪

• 批准号: 543577782
• 金额: --
• 依托单位: Albert-Ludwigs-Universität Freiburg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2024
• 资助国家: 德国
• 起止时间: 2023-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/543577782?language=en
• 关键词: Fast; hybrid; mass; spectrometer; complexome

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.

所要求的timsTOF HT质谱仪将取代申请人迄今为止使用的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II研究所研究了质膜上和跨质膜的快速信号转导的分子基础，特别是哺乳动物大脑中的神经元。这种信号转导的空间和时间精确性背后的核心组织原理是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，并成功地将其应用于一系列离子通道，膜受体和转运蛋白。实验工作流程包括（i）从天然组织中分离膜组分，（ii）亲和纯化靶蛋白（多表位AP-MS，“meAP-MS”），（iii）蛋白质级分的高分辨分离和膜亚分级（复合体分析或细胞器蛋白质组学），和（iv）用于鉴定和定量蛋白质的高分辨率纳米-HPLC偶联质谱法，包括其后-翻译修饰如磷酸化、烷基化或脂质修饰。因此，它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/循环速度方面的高性能质谱法，特别是对于高分辨率络合物组分析（通过该研究所最近开发的“低温切片蓝色天然MS”，csBN-MS技术），其需要具有高分析覆盖率的扩展测量系列（每个生物样品200-400次测量）。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，> 5倍，性能仍然上级）和在扩展的动态范围内的定量（即，获得大量蛋白质/肽的定量数据，覆盖单个测量内的4-5个数量级），该仪器是理想的。这种高分辨率的复合物组分析使第一次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，购买timsTOF HT对申请人团队未来的工作/项目至关重要。