Advances in mass spectrometry for biological science

The main advantages of using mass spectrometry for biological science, as compared with other analytical or structural methods, are (i) its ability to treat a very wide range of biomolecules and (ii) the extreme sensitivity that often goes down to only a few hundred molecules of sample. Following the ionization of the molecules of interest, information on their composition and structure is obtained through the determination of absolute masses of a set of ion fragments produced by dissociation. Mass spectrometry is thus a key method for identifying and quantifying biomolecules and their modifications. In parallel with the expanding number of completely sequenced genomes, proteomics, metabolomics and lipidomics aim at a comprehensive and quantitative analysis of a wide array of expressed proteins, metabolites and lipids, respectively, in biological samples. Mass spectrometry is an invaluable tool that enables such an analysis in high throughput. Accurate and unbiased information about changes of thousands of biomolecules constitutes a sizable fraction of an instantaneous snapshot of the entire physiology of a cell. Biomarkers can then be determined and possibly lead to a possible distinction between normal and diseased states of the cell for the purpose of pharmaceutical drug development or early medical diagnosis.

The mass-spectrometry tool evolves through investigations that are conducted in a wide variety of research fields. From the instrumental point of view, besides the design of new high-resolution and large dynamical mass-range spectrometers, the most popular production methods of gas-phase biomolecular ions, i.e. electrospray ionization (ESI) and matrix-assisted laser desorption–ionization (MALDI), present problems in the case of membrane proteins and fragile assemblies of weakly-bound biomolecules. Recently developed techniques bring into reality native mass-spectrometry that is capable of isolating non-covalent complexes directly from membranes, viruses or cells without modifying or removing any constituent used to generate high-order structures. The obtained topological information helps to decipher intricate interaction network.

Once the exact mass of biomolecular ions is measured, determination of the sequence of their elementary building blocks and their possible modifications (e.g. sequence and post-translational modifications of a protein) proceeds through successive dissociation and identification of the fragments with the help of databanks storing fragment mass fingerprints. In addition to the well-established low-energy collision-induced dissociation (CID), other fragmentation processes such as electron capture dissociation (ECD) have been introduced and provide different bond-breaking patterns. Understanding the corresponding dissociation mechanisms requires high-level quantum chemistry. It is hoped that not only qualitative but also quantitative prediction of fragmentation pathways will lead to improved interpretation of observed fragmentation patterns.

Following the compositional identification of a biomolecular ion, determination of its conformational and chemical structure is the next step, often achieved with the help of structural tools such as hydrogen/deuterium (H/D) exchange, ion mobility (IM) and gas-phase infrared spectroscopy techniques. With the advent of widely-tunable and high-power infrared sources in the form of either free-electron lasers or table-top optical parametric oscillators (OPOs), vibrational spectroscopy of a small amount of ions stored in high-resolution mass-spectrometers can be accomplished. Infrared multiphoton dissociation (IRMPD) is a quasi-universal technique providing rather low-resolution (typically 10–20 cm⁻¹) spectra but nevertheless quite sufficient for the determination of structures of small isolated biomolecular ions or their charged fragments. Conformers and tautomers are identified through comparison between experimentally obtained spectra and those predicted from modelling. When the size of gas-phase ions increases, too many conformations may coexist at room temperature, which often yields heavily congested spectra. Disentanglement of those spectra is facilitated by the use of ion-mobility measurements assessing the number of conformers present. This situation is rapidly changing with the advent of spectroscopy of mass-selected biomolecular ions stored at very low (<10 K) temperatures.

The present Themed Issue collects studies taking advantage of different experimental approaches on a wide scale of masses ranging from very small molecular systems (such as amino acids or drugs) to peptides, oligonucleotides and carbohydrates, and even up to entities as large as ATPases or virus capsids. The question of the relationship between structures of isolated biomolecular ions issued from ESI sources and their corresponding solution-phase structures receives answers from spectroscopic studies conducted on confined ions with IRMPD. Oligonucleotides adopt a variety of secondary structures. While guanine-rich sequences adopt quadruplex structures, cytosine-rich sequences form less investigated intercalated duplexes (i-motif). The combined IRMPD and ion mobility studies of Rosu et al. (DOI: 10.1039/c0cp00782j) show that i-motifs are preserved in the gas phase in the case of low-charge anions. Structures of the biologically active S-nitrosocaptopril and its parent species lacking NO are presented by Coletti et al. (DOI: 10.1039/c0cp00671h) and open perspectives for understanding S-nitrosation and the role of NO in cells.

Metals play crucial roles in proteins, in particular in enzymes. The intimate structural details of the binding of alkali and larger metal ions to phenylalanine are revealed through IRMPD by Dunbar et al. (DOI: 10.1039/c0cp00784f). Binding of metals to cystein-rich proteins plays pivotal roles. Ye and Armentrout relate experimental and predicted absolute binding energies of water molecules to sodiated cysteine cations and determine the size of the first hydration shell (DOI: 10.1039/c0cp00302f). The release of zinc from the large metallothionein protein towards suitable acceptors is investigated by means of native mass spectrometry with the help of NMR structures by Leszczyszyn and Blindauer (DOI: 10.1039/c0cp00680g).

Post-translational modifications of proteins modulate their activity and the determination of their existence and of their position constitutes a required step in proteomics. Andreazza and Bowie (DOI: 10.1039/c0cp00717j) show how negative ion cleavage by means of CID identifies and localizes intra- and inter-disulfide bonds. Information obtained by ECD complements that from CID. However, the mechanism from which different fragmentation patterns result is still a matter of debate. Among them, charge-reduction of protonated sites and H atom transfer have been proposed. Gregersen and Tureček (DOI: 10.1039/c0cp00597e) use neutralization–reionization mass spectrometry to investigate bond cleavage in radical intermediates produced by electron capture from protonated ions. Together with this mechanism, other proposed ECD mechanisms are examined in nitrated peptides in a comprehensive way by Jones and Cooper (DOI: 10.1039/c0cp00623h). Some cleavage processes such as ECD or electron transfer dissociation (ETD) can lead to a homogeneous ensemble of fragments while others such as high-energy collisional dissociation (HCD) or CID exhibit statistical patterns. Considering a very large dataset of tryptic peptide spectra, Good et al. (DOI: 10.1039/c0cp00514b) investigate the role of the high energy content of ions activated by collisional processes.

Coupling of ion mobility to mass spectrometry (IM-MS) can provide detailed structural information and relative abundances of different conformers. This is crucial in the difficult case of glycans that are not linear like peptides but rather exhibit complex branching patterns.

Preserving large and fragile protein complexes in solution or cells intact while they are transferred to the gas phase towards a spectrometer is a difficult task. Hogan et al. (DOI: 10.1039/c0cp01208d) employ the combination of a differential mobility analyzer and a mass spectrometer to investigate the simultaneous use of supercharging and charge-reduction agents in the case of a 97 kDa protein and its multimers. This increases the charge-state distributions of electrosprayed complex ions, which facilitates mass analysis but not at the expense of non-denaturating conditions. Analysis of membrane protein complexes represents a challenging problem. The complex subunit stoichiometries of different ATP synthases are determined by Hoffmann et al. (DOI: 10.1039/c0cp00733a) using a novel ion desorption method relying on laser explosion of microdroplets gently delivering large masses and very low charge state ions. Differences and conserved features in those bacterial and eukaryotic enzymes are revealed.

Virus capsids are fascinating objects whose role is protection of the virus genome. The capsid construction relies on self-assembling of small coat protein sub-units. Design of new therapeutic solutions with cargos capable of vehiculating and delivering drugs takes advantage of a detailed understanding of the capsid container building process. With the help of H/D exchange, Morton et al. (DOI: 10.1039/c0cp00817f) were able to locate regions of a protein dimer influenced by the binding of a genomic single-strand RNA stem-loop that intervene in the initiation of capsid assembling. Investigating the hepatitis B virus, Uetrecht et al. (DOI: 10.1039/c0cp00692k) scrutinize with native mass spectrometry how capsids that are stable assemblies can nevertheless dynamically offer to internally enclosed species the possibility of interacting with their outside environment.

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