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

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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.