Simultaneous probing of primary and higher-order protein structure by native and top-down mass spectrometry

通过天然和自上而下的质谱法同时探测初级和高级蛋白质结构

• 批准号: 524226614
• 负责人: Professor Dr. Frederik Lermyte
• 金额: --
• 依托单位: Arbeitsgruppe Conformation-Sensitive Mass Spectrometry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524226614?language=en
• 关键词: Simultaneous; probing; primary; higher; order

To understand the function of biomolecules, particularly proteins, it is imperative to understand their 3D structure and interactions. This field is known as structural biology, and has traditionally relied on methods such as X-ray crystallography, nuclear magnetic resonance, and more recently, cryo-electron microscopy. These methods, while powerful, have their limitations; for example, limited sensitivity to differentiate between protein variants, non-trivial sample preparation, and difficulty to simultaneously characterise conformations that co-exist and/or interconvert over time. Simultaneous characterisation of protein sequence, conformation, and of the formation and stoichiometry of noncovalent complexes therefore represents an important bottleneck in biological and medical research. Mass spectrometry (MS) can characterise both the sequence and conformation of proteins, as well as their assembly into complexes. With appropriate sample preparation, it is nearly universally applicable, and can probe timescales from milliseconds to days. The Lermyte lab at TU Darmstadt has state-of-the-art equipment as well as a track record in so-called 'native' mass spectrometry of protein complexes. We also have considerable expertise with the use of advanced fragmentation methods to study the sequence of intact proteins, and we are at the heart of several collaborative networks, both locally and spanning different continents. Using a unique set of model proteins, we will develop new methods that combine native ionisation with gas-phase fragmentation and ion mobility measurements. These approaches will allow us to study protein primary structure, folding, complex stoichiometry, and unfolding pathways. We will correlate our results to published protein structures from classical methods, as well as to orthogonal solution-phase labelling experiments carried out in our lab. Additionally, we will investigate the use of solution additives (low-volatility co-solvents, metal complexes, and metal cations) to modulate the movement of charge carriers (protons and electrons) during and after electrospray ionisation. This movement alters the fragmentation of peptides and proteins, potentially leading to more extensive fragmentation and better characterisation of the primary structure. This project will therefore result in improvements in the MS-based characterisation of all levels of protein structure.

为了了解生物分子的功能，特别是蛋白质，必须了解它们的3D结构和相互作用。这个领域被称为结构生物学，传统上依赖于X射线晶体学，核磁共振和最近的低温电子显微镜等方法。这些方法虽然强大，但也有其局限性;例如，区分蛋白质变体的灵敏度有限，样品制备不简单，以及难以同时检测共存和/或随时间相互转化的构象。因此，同时表征蛋白质序列、构象以及非共价复合物的形成和化学计量是生物学和医学研究中的一个重要瓶颈。质谱（MS）可以分析蛋白质的序列和构象，以及它们组装成复合物。通过适当的样品制备，它几乎是普遍适用的，可以探测从毫秒到几天的时间尺度。达姆施塔特工业大学的Lermyte实验室拥有最先进的设备，并在蛋白质复合物的所谓“天然”质谱方面有着良好的记录。我们还拥有使用先进的片段化方法研究完整蛋白质序列的丰富专业知识，我们是本地和跨越不同大陆的几个合作网络的核心。使用一套独特的模型蛋白质，我们将开发新的方法，结合联合收割机本地电离气相碎片和离子迁移率测量。这些方法将使我们能够研究蛋白质的一级结构，折叠，复杂的化学计量学和展开途径。我们将把我们的结果与经典方法中发表的蛋白质结构以及我们实验室进行的正交溶液相标记实验相关联。此外，我们将研究使用溶液添加剂（低挥发性共溶剂、金属配合物和金属阳离子）来调节电喷雾电离期间和之后电荷载体（质子和电子）的运动。这种运动改变了肽和蛋白质的片段化，可能导致更广泛的片段化和更好的一级结构表征。因此，该项目将导致基于MS的所有水平的蛋白质结构表征的改进。