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

要了解生物分子，特别是蛋白质的功能，必须了解它们的三维结构和相互作用。这个领域被称为结构生物学，传统上依赖于x射线晶体学、核磁共振和最近的冷冻电子显微镜等方法。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，非琐碎样品制备，以及难以同时表征共存和/或随时间相互转换的构象。因此，蛋白质序列、构象以及非共价复合物的形成和化学计量学的同时表征是生物和医学研究中的一个重要瓶颈。质谱（MS）可以表征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，并且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勒米特实验室拥有最先进的设备，在所谓的“天然”蛋白质复合物质谱分析方面也有良好的记录。我们在使用先进的碎片化方法来研究完整蛋白质序列方面也有相当的专业知识，我们是几个合作网络的核心，包括本地和跨越不同大陆。使用一组独特的模型蛋白，我们将开发结合天然电离和气相破碎和离子迁移率测量的新方法。这些方法将使我们能够研究蛋白质的初级结构、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从经典方法中发表的蛋白质结构以及在我们实验室进行的正交溶液标记实验联系起来。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）来调节电荷载流子（质子和电子）在电喷雾电离期间和之后的运动。这种运动改变了多肽和蛋白质的碎片化，可能导致更广泛的碎片化和更好的初级结构表征。因此，该项目将改进基于质谱的各级蛋白质结构表征。

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.