Cross‐Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three‐dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X‐ray crystallography. Structural MS unites different MS‐based techniques, such as hydrogen/deuterium exchange, native MS, ion‐mobility MS, protein footprinting, and chemical cross‐linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross‐linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross‐linking/MS is currently one of the most promising MS‐based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.     

Metal displacement and stoichiometry of protein‐metal complexes under native conditions using capillary electrophoresis/mass spectrometry

Increases in the study of protein‐metal complexes, as well as in metal displacement in protein‐metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein‐metal complexes. Both nuclear magnetic resonance (NMR) and X‐ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI‐MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein‐metal complexes. However, since protein‐metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI‐MS analysis requires off‐line sample clean‐up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non‐specific salt adduct formation by the protein‐metal complexes under ESI‐MS, which complicates the identification and stoichiometry measurements of the protein‐metal complexes. Because of the high mass of protein‐metal complexes and lack of sufficient resolution by most mass spectrometers to separate non‐specific from specific metal‐protein complexes, accurate protein‐metal stoichiometry measurements require some form of sample clean up prior to ESI‐MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium‐resolution (～10 000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein‐metal complexes under physiological conditions (pH ～7). The metal displacement of Co²⁺ to Cd²⁺, two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept. Copyright © 2010 John Wiley & Sons, Ltd.     

The Application of Fluorine‐Containing Reagents in Structural Proteomics

Structural proteomics refers to large‐scale mapping of protein structures in order to understand the relationship between protein sequence, structure, and function. Chemical labeling, in combination with mass‐spectrometry (MS) analysis, have emerged as powerful tools to enable a broad range of biological applications in structural proteomics. The key to success is a biocompatible reagent that modifies a protein without affecting its high‐order structure. Fluorine, well‐known to exert profound effects on the physical and chemical properties of reagents, should have an impact on structural proteomics. In this Minireview, we describe several fluorine‐containing reagents that can be applied in structural proteomics. We organize their applications around four MS‐based techniques: a) affinity labeling, b) activity‐based protein profiling (ABPP), c) protein footprinting, and d) protein cross‐linking. Our aim is to provide an overview of the research, development, and application of fluorine‐containing reagents in protein structural studies.     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

Current limitations in native mass spectrometry based structural biology

Nowadays, mass spectrometry plays an important role in structural biology. At one end it can be used to investigate intact protein complexes, providing details about the complex composition, topology, stability, and dynamics, whereas at the other end the protein’s identity and possible modifications can be visualized using proteomics approaches. Combining all this information allows the generation of detailed models for functional biological assemblies. Here, a perspective on the application of native mass spectrometry in structural biology is presented. The potential of this technique and some important current limitations are discussed. This includes issues regarding the quality/homogeneity of the sample, the dissociation efficiency of protein complexes during tandem mass spectrometric analysis, and some boundaries of ion mobility mass spectrometry.     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.     

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein‐based pharmaceuticals. The introduction and maturation of “soft” ionization methods, such as electrospray ionization and matrix‐assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis. This review summarizes these challenges and provides an overview of the state‐of‐the‐art in sample preparation and fractionation of proteins for mass spectrometric analysis, with an emphasis on those used for top‐down proteomic approaches. Biological fluids, particularly important for clinical and pharmaceutical applications and their characteristics are also discussed. While immunoaffinity‐based methods are addressed, more attention is given to non‐immunoaffinity‐based methods, such as precipitation, coacervation, size exclusion, dialysis, solid‐phase extraction, and electrophoresis. These techniques are presented in the context of a significant number of studies where they have been developed and utilized.     

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.     

Liberating Native Mass Spectrometry from Dependence on Volatile Salt Buffers by Use of Gábor Transform

Volatile salts, such as ammonium acetate, are commonly used in buffers for the analysis of intact proteins and protein complexes in native electrospray ionization mass spectrometry. Although these solutions are not technically buffers near pH 7, the volatile nature of the salt minimizes ion adduction to proteins upon transfer to vacuum. Conversely, common biochemical salt buffers, such as Tris/NaCl, are not traditionally used in native mass spectrometry because of the tendency of sodium and other ions to adduct to proteins or form large cluster ions, severely frustrating accurate mass assignment. Here, we demonstrate a Gábor transform method for extracting signal from native-like protein ions even in the presence of a large salt-cluster background. We further show the utility of this method in characterizing polymers and show that the measured average mass of long-chain polyethylene glycol ions from a commercial polymer sample is ∼30 % higher than the manufacturer-estimated average mass. It is expected that this method will enable more widespread use of conventional biochemical buffers in native mass spectrometry and decrease dependence on volatile salts.     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

Adding a new separation dimension to MS and LC–MS: What is the utility of ion mobility spectrometry?

Ion mobility spectrometry is an analytical technique known for more than 100 years, which entails separating ions in the gas phase based on their size, shape, and charge. While ion mobility spectrometry alone can be useful for some applications (mostly security analysis for detecting certain classes of narcotics and explosives), it becomes even more powerful in combination with mass spectrometry and high‐performance liquid chromatography. Indeed, the limited resolving power of ion mobility spectrometry alone can be tackled when combining this analytical strategy with mass spectrometry or liquid chromatography with mass spectrometry. Over the last few years, the hyphenation of ion mobility spectrometry to mass spectrometry or liquid chromatography with mass spectrometry has attracted more and more interest, with significant progresses in both technical advances and pioneering applications. This review describes the theoretical background, available technologies, and future capabilities of these techniques. It also highlights a wide range of applications, from small molecules (natural products, metabolites, glycans, lipids) to large biomolecules (proteins, protein complexes, biopharmaceuticals, oligonucleotides).     

Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study

Can the structures of small to medium‐sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion‐mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β‐lactoglobulin, which are prototypical examples of helical and β‐sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.     

Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques.

Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction ( approximately 20%) retaining the initial methionine. By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E. coli grown in different media. Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used. In particular, this technique was applied to identify and quantify fatty acids bound to CRBP. Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein. Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression.     

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.     

The Evolving Contribution of Mass Spectrometry to Integrative Structural Biology

Protein complexes are key catalysts and regulators for the majority of cellular processes. Unveiling their assembly and structure is essential to understanding their function and mechanism of action. Although conventional structural techniques such as X-ray crystallography and NMR have solved the structure of important protein complexes, they cannot consistently deal with dynamic and heterogeneous assemblies, limiting their applications to small scale experiments. A novel methodological paradigm, integrative structural biology, aims at overcoming such limitations by combining complementary data sources into a comprehensive structural model. Recent applications have shown that a range of mass spectrometry (MS) techniques are able to generate interaction and spatial restraints (cross-linking MS) information on native complexes or to study the stoichiometry and connectivity of entire assemblies (native MS) rapidly, reliably, and from small amounts of substrate. Although these techniques by themselves do not solve structures, they do provide invaluable structural information and are thus ideally suited to contribute to integrative modeling efforts. The group of Brian Chait has made seminal contributions in the use of mass spectrometric techniques to study protein complexes. In this perspective, we honor the contributions of the Chait group and discuss concepts and milestones of integrative structural biology. We also review recent examples of integration of structural MS techniques with an emphasis on cross-linking MS. We then speculate on future MS applications that would unravel the dynamic nature of protein complexes upon diverse cellular states.

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Ultrafast Structural Fluctuations of Myoglobin‐Bound Thiocyanate and Selenocyanate Ions Measured with Two‐Dimensional Infrared Photon Echo Spectroscopy

Structural dynamics within the distal cavity of myoglobin protein is investigated using 2D‐IR and IR pump–probe spectroscopy of the N≡C stretch modes of heme‐bound thiocyanate and selenocyanate ions. Although myoglobin‐bound thiocyanate group shows a doublet in its IR absorption spectrum, no cross peaks originating from chemical exchange between the two components are observed in the time‐resolved 2D IR spectra within the experimental time window. Frequency–frequency correlation functions of the two studied anionic ligands are obtained by means of a few different analysis approaches; these functions were then used to elucidate the differences in structural fluctuation around ligand, ligand–protein interactions, and the degree of structural heterogeneity within the hydrophobic pocket of these myoglobin complexes.     

Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Comparative investigations of the secondary ion emission of metal complexes under MeV and keV ion bombardment

A series of ionic and neutral Group VIII transition metal complexes with molecular masses up to 2500 u were analysed by time-of-flight secondary ion mass spectrometry (SIMS) and plasma desorption mass spectrometry (PDMS). The secondary ion emission, the secondary ion yields and the yield ratios Y(PDMS)/Y(SIMS) of 20 ionic and neutral metal complexes were determined. Both techniques generally provide both molecular and fragment ion information. Characteristic fragmentation patterns give useful data for structural characterization. Additionally, the stabilities of different secondary ion species were compared by their half-lives. Both PDMS and SIMS are very sensitive, yielding optimum spectra from total sample sizes as low as 5 nmol, and the sample consumption is negligible.     

Native Mass Spectrometry from Common Buffers with Salts That Mimic the Extracellular Environment

Nonvolatile salts are essential for the structures and functions of many proteins and protein complexes but can severely degrade performance of native mass spectrometry by adducting to protein and protein complex ions, thereby reducing sensitivity and mass measuring accuracy. Small nanoelectrospray emitters are used to form protein and protein complex ions directly from high‐ionic‐strength (>150 mm ) nonvolatile buffers with salts that mimic the extracellular environment. Charge‐state distributions are not obtained for proteins and protein complexes from six commonly used nonvolatile buffers and ≥150 mm Na⁺ with conventionally sized nanoelectrospray emitter tips but are resolved with 0.5 μm tips. This method enables mass measurements of proteins and protein complexes directly from a variety of commonly used buffers with high concentrations of nonvolatile salts and eliminates the need to buffer exchange into volatile ammonium buffers traditionally used in native mass spectrometry.