Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Chemical investigations of herbarium material for alkaloids

The chemical and biological screening of plants is reviewed briefly as an introduction to the concept of the use of herbarium material for investigations into the chemical constituents of plants. Such materials have been examined for a wide range of chemicals, and in particular, examples of the extraction of alkaloids from species of Rubiaceae and Papaveraceae are discussed. The present state of the art of such physical techniques as mass and nuclear magnetic resonance spectrometry for these investigations is commented upon and the plea is made that, wherever possible, wider use be made of herbarium material for chemical studies.     

Purifying protein complexes for mass spectrometry: applications to protein translation

Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.     

Use of atmospheric pressure ionization mass spectrometry in enantioselective liquid chromatography

Recent advances in mass spectrometry have rendered it an attractive and versatile tool in industrial and academic research laboratories. As a part of this rapid growth, a considerable body of literature has been devoted to the application of mass spectrometry in studies involving enantioselectivity, molecular recognition, and supramolecular chemistry. In concert with separation techniques such as capillary electrophoresis and liquid chromatography, mass spectrometry allows rapid characterization of a large array of molecules in complex mixtures. A majority of these findings have been made possible by the introduction of 'soft-ionization' techniques such as electrospray ionization interface. Other techniques such as atmospheric pressure chemical ionization mass spectrometry have been widely used as a rugged interface for quantitative liquid chromatography-mass spectrometry. Herein, we present a brief overview of the above techniques accompanied with several examples of enantioselective capillary electrophoresis- and liquid chromatography-mass spectrometry in drug discovery and development. Although the emphasis of this article is on quantitative enantiomeric chromatography-mass spectrometry, we envisage that similar strategies are adaptable in qualitative studies.     

Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination

Mass spectrometry is now established as a powerful tool for the study of the stoichiometry, interactions, dynamics, and subunit architecture of large protein assemblies and their subcomplexes. Recent evidence has suggested that the 3D structure of protein complexes can be maintained intact in the gas phase, highlighting the potential of ion mobility to contribute to structural biology. A key challenge is to integrate the compositional and structural information from ion mobility mass spectrometry with molecular modelling approaches to produce 3D models of intact protein complexes. In this review, we focus on the mass spectrometry of protein-nucleic acid assemblies with particular attention to the application of ion mobility, an emerging technique in structural studies. We also discuss the challenges that lie ahead for the full integration of ion mobility mass spectrometry with structural biology.     

Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination

Mass spectrometry is now established as a powerful tool for the study of the stoichiometry, interactions, dynamics, and subunit architecture of large protein assemblies and their subcomplexes. Recent evidence has suggested that the 3D structure of protein complexes can be maintained intact in the gas phase, highlighting the potential of ion mobility to contribute to structural biology. A key challenge is to integrate the compositional and structural information from ion mobility mass spectrometry with molecular modelling approaches to produce 3D models of intact protein complexes. In this review, we focus on the mass spectrometry of protein-nucleic acid assemblies with particular attention to the application of ion mobility, an emerging technique in structural studies. We also discuss the challenges that lie ahead for the full integration of ion mobility mass spectrometry with structural biology.     

Chemical cross-linking and native mass spectrometry: A fruitful combination for structural biology

Mass spectrometry (MS) is becoming increasingly popular in the field of structural biology for analyzing protein three-dimensional-structures and for mapping protein–protein interactions. In this review, the specific contributions of chemical crosslinking and native MS are outlined to reveal the structural features of proteins and protein assemblies. Both strategies are illustrated based on the examples of the tetrameric tumor suppressor protein p53 and multisubunit vinculin-Arp2/3 hybrid complexes. We describe the distinct advantages and limitations of each technique and highlight synergistic effects when both techniques are combined. Integrating both methods is especially useful for characterizing large protein assemblies and for capturing transient interactions. We also point out the future directions we foresee for a combination of in vivo crosslinking and native MS for structural investigation of intact protein assemblies.     

Structural Genomics of Eukaryotic Targets at a Laboratory Scale

Structural genomics programs are distributed worldwide and funded by large institutions such as the NIH in United-States, the RIKEN in Japan or the European Commission through the SPINE network in Europe. Such initiatives, essentially managed by large consortia, led to technology and method developments at the different steps required to produce biological samples compatible with structural studies. Besides specific applications, method developments resulted mainly upon miniaturization and parallelization. The challenge that academic laboratories faces to pursue structural genomics programs is to produce, at a higher rate, protein samples. The Structural Biology and Genomics Department (IGBMC – Illkirch – France) is implicated in a structural genomics program of high eukaryotes whose goal is solving crystal structures of proteins and their complexes (including large complexes) related to human health and biotechnology. To achieve such a challenging goal, the Department has established a medium-throughput pipeline for producing protein samples suitable for structural biology studies. Here, we describe the setting up of our initiative from cloning to crystallization and we demonstrate that structural genomics may be manageable by academic laboratories by strategic investments in robotic and by adapting classical bench protocols and new developments, in particular in the field of protein expression, to parallelization.     

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

Visualizing transiently associated catalytic domains in assembly-line biosynthesis using cryo-electron microscopy

While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein–protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure–function relationships. Here, we outline examples in which transient protein–protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein–protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein–protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.     

Errata

Abstract

Mass spectrometry (MS)-based proteomics is an unrivaled tool for studying complex biological systems and diseases in the post-genomic era. In recent years, MS has emerged as a powerful structural biological tool to characterize protein conformation and conformational dynamics. The advantages of MS in structural studies are most evident for membrane proteins such as GPCRs (G protein-coupled receptors), where other well-established structural methods such as X-ray crystallography and NMR remain challenging. For proteins with available high-resolution structures, MS-based structural strategies can provide valuable, previously inaccessible information on protein conformational changes and dynamics, protein motion/flexibility, ligand–protein binding, and protein–protein interfaces. In the past several years, we have developed and adapted a number of MS-based structural approaches, such as CDSiL-MS (Conformational changes and Dynamics using Stable-isotope Labeling and MS), CXMS (Crosslinking/MS) and HDXMS (Hydrogen-Deuterium Exchange MS), to study protein structures and conformational dynamics in human β2-adrenegic receptor (β2AR) signaling. In this mini-review, we will highlight several examples demonstrating the power of MS in structural analysis to better elucidate the structural basis of GPCR signaling, particularly through the β-arrestin-mediated GPCR signaling pathway.     

Native mass spectrometry for understanding dynamic protein complex

Biomolecules have evolved to perform specific and sophisticated activities in a highly coordinated manner organizing into multi-component complexes consisting of proteins, nucleic acids, cofactors or ligands. Understanding such complexes represents a task in earnest for modern bioscience. Traditional structural techniques when extrapolating to macromolecules of ever increasing sizes are confronted with limitations posed by the difficulty in enrichment, solubility, stability as well as lack of homogeneity of these complexes. Alternative approaches are therefore prompted to bridge the gap, one of which is native mass spectrometry. Here we demonstrate the strength of native mass spectrometry, used alone or in combination with other biophysical methods such as analytical ultracentrifugation, small-angle neutron scattering, and small-angle X-ray scattering etc., in addressing dynamic aspects of protein complexes including structural reorganization, subunit exchange, as well as the assembly/disassembly processes in solution that are dictated by transient non-covalent interactions. We review recent studies from our laboratories and others applying native mass spectrometry to both soluble and membrane-embedded assemblies. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

Engineering chromatin states: Chemical and synthetic biology approaches to investigate histone modification function

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

Ion mobility–mass spectrometry for structural proteomics

Ion mobility coupled to mass spectrometry has been an important tool in the fields of chemical physics and analytical chemistry for decades, but its potential for interrogating the structure of proteins and multiprotein complexes has only recently begun to be realized. Today, ion mobility–mass spectrometry is often applied to the structural elucidation of protein assemblies that have failed high-throughput crystallization or NMR spectroscopy screens. Here, we highlight the technology, approaches and data that have led to this dramatic shift in use, including emerging trends such as the integration of ion mobility–mass spectrometry data with more classical (e.g., ‘bottom-up’) proteomics approaches for the rapid structural characterization of protein networks.     

Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

Lantibiotics and microcins: polypeptides with unusual chemical diversity

Bacterial-derived antimicrobial polypeptides enjoy a large degree of structural and chemical diversity. Two well-studied examples of such polypeptides are the lanthionine-containing lantibiotics produced by a variety of Gram-positive bacteria, and their Gram-negative counterparts, the microcins. Both groups are produced as gene-encoded precursor peptides and undergo post-translational modification to generate the active moieties. Structure elucidation of novel lantibiotics and microcins has recently uncovered further novel structural and chemical features and, combined with the generation of analogue peptides by genetic manipulation, new insights into structure-function relationships have been gained. Furthermore, study of the mode of action of the lantibiotics nisin and mersacidin has revealed their use of a 'docking molecule' in the target cell to facilitate their biological activities. Meanwhile, in vitro studies with microcin B17 have helped to uncover the molecular mechanisms by which post-translational modification results in the formation of heterocyclic oxazole and thiazole rings. From a practical standpoint, both groups of polypeptides represent new lead structures for future development of antimicrobial agents, whilst the identification of the 'docking molecules' represents a step forward in the search for novel targets for future antibiosis.     

Native mass spectrometry: a bridge between interactomics and structural biology

Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.     

Investigation of protein-RNA interactions by mass spectrometry--Techniques and applications

Protein-RNA complexes play many important roles in diverse cellular functions. They are involved in a wide variety of different processes in growth and differentiation at the various stages of the cell cycle. As their function and catalytic activity are directly coupled to the structural arrangement of their components--proteins and ribonucleic acids--the investigation of protein-RNA interactions is of great functional and structural importance. Here we discuss the most prominent examples of protein-RNA complexes and describe some frequently used purification strategies. We present various techniques and applications of mass spectrometry to study protein-RNA complexes. We discuss the analysis of intact complexes as well as proteomics-based and crosslinking-based approaches in which proteins are cleaved into smaller peptides. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.