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Since the sequencing of the human genome nearly twenty years ago, it has been noted that the relatively limited number of genes comprising it – 20 000 or so – seems at odds with our observed biological complexity. Indeed, today it is commonly understood that this underestimates the number of functional protein products present in a human organism by several orders of magnitude. This is due to RNA splicing, post-translational modification, formation of noncovalent functional complexes, and so on. To further appreciate the complexity of biological systems, the presence of other classes of biomolecules that are not as clearly associated with a particular gene as proteins also needs to be taken into account, for example polysaccharides and lipids. It is clear, therefore, that structural characterisation needs to happen on the level of functional biomolecules, and not just that of DNA or RNA, which are primarily information carriers.

Mass spectrometry has emerged as the method of choice for the fast, high-throughput analysis of complex mixtures of biomolecules, and this is in large part due to the ability of this technique to break down ions in the gas phase and measure the mass of the resulting fragments, yielding a wealth of structural information. While the initial focus was on the determination of protein and peptide sequence – mostly relying on collision-induced dissociation of ions – in recent years there has been a tremendous expansion of the application field of mass spectrometry, both to other classes of biomolecules, and to the characterisation of higher-order rather than just primary structure. To a large extent, this evolution has been driven by technological developments in the available fragmentation methods. Given the extraordinary pace of these developments in recent years, information tends to be somewhat scattered across the literature, without a single, authoritative guide being available. This book aims to address this issue by presenting a comprehensive overview of advanced fragmentation methods in biomolecular mass spectrometry and recent developments therein.

In Chapter 1, some key concepts in modern mass spectrometry will be briefly recapitulated, although a degree of familiarity with this material is assumed. Chapter 2 discusses two of the most developed and widely used advanced fragmentation methods, electron capture and electron transfer dissociation, along with their mechanism and different ways of implementing them in practice. Chapter 3 follows up on this, and focuses on the use of these two methods for the determination of the primary structure of peptides and intact proteins. In Chapters 4 and 5, the focus is still on proteins, but now on determining higher-order structure. Chapter 4 discusses how methods such as hydrogen–deuterium exchange and chemical crosslinking benefit from advanced fragmentation methods. In these methods, chemical modification of the protein occurs in a conformation-sensitive manner, but the fragmentation step is usually performed after chemical denaturation of the protein, i.e., annihilation of higher-order structure. Chapter 5 explores approaches in which an unmodified peptide or protein is first transferred into the gas phase in a way that preserves at least some degree of the higher-order structure, and then subjected to electron-based dissociation. Specifically, the chapter elaborates on how this residual structure affects the fragmentation pattern, and how this results in structural details being revealed through careful analysis of the fragment spectrum. While most peptides and proteins are efficiently ionised in positive ion mode, Chapter 6 discusses electron detachment dissociation and negative electron transfer dissociation, two activation methods that have been developed to fragment negative ions, which are useful for oligonucleotides, oligosaccharides, and proteins with acidic post-translational modifications. Chapter 7 also focuses on anions, and specifically on a fragmentation method called electron photodetachment dissociation. Turning momentarily back to peptides and proteins, Chapter 8 discusses somewhat more exotic alternatives to electron-based dissociation for using gas-phase radical chemistry to induce bond-selective fragmentation, including radical-directed dissociation and photoelectron transfer dissociation. Lipids are often neglected in mass spectrometry textbooks, as they constitute a challenging class of analytes. Significant developments have occurred in recent years, however, and the biological importance of these biomolecules is not to be underestimated. For this reason, Chapter 9 is devoted to advanced tandem MS methods for determination of lipid structure. Chapter 10 discusses photon-based methods for ion activation, with particular attention to infrared multiphoton photodissociation and ultraviolet photodissociation. Finally, Chapter 11 quite literally investigates what happens when ions run into a wall, and as it turns out, activating biomolecular ions by having them collide with a surface leads to interesting fragmentation behaviour that can provide details on both primary and higher-order structure.

This book was not realised overnight. It took slightly over a year to go from the initial discussions with the Royal Society of Chemistry to writing this preface. Even then, this book could not have been realised at all without the tremendous efforts of all contributing authors, to whom I am immensely grateful. It has been delightful to see the willingness of this diverse group of some of the world's leading experts in their respective areas to share some of their accrued expertise in this book. I also thank the Royal Society of Chemistry, and specifically Janet Freshwater, Liv Towers, and Katie Morrey, for their editorial support. In a broader sense, I am grateful to the entire biomolecular mass spectrometry community for driving the continuous developments that this exciting field has seen so far, and will undoubtedly continue to see in the future.

Frederik Lermyte

Technische Universität Darmstadt

Darmstadt, July 2020

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