Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Review: applications of mass spectrometry techniques in the investigation of milk proteome

The introduction of "soft" desorption/ionization methods such as electrospray ionization and matrix-assisted laser desorption/ionization has determined a breakthrough in the application of mass spectrometry to the structural analysis of proteins. The contemporary advancement of bioinformatics, together with the possibility to combine these mass spectrometric methods with electrophoretic or chromatographic separation techniques has opened up the new field of proteome analysis and, more generally, has established these approaches as indispensable tools for protein and peptide analysis in complex mixtures, such as milk and milk- derived foods. Here, a necessarily not exhaustive series of current applications of mass spectrometry-based techniques for the characterization of milk proteins will be summarized. These include the characterization of milk protein polymorphism, determination of the structural modifications induced on milk proteins by industrial processes, investigation of milk adulterations and characterization of milk allergens.     

Principles of Fourier transform ion cyclotron resonance mass spectrometry and its application in structural biology

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has become increasingly significant within recent years. The inherently ultra-high resolution and mass accuracy allow unequivocal assignments of chemical formulae to be made and further structural elucidation can be conducted through the utilization of tandem mass spectrometry techniques. With the advent of electrospray ionization (ESI), FT-ICR mass spectrometry has become a powerful tool for the investigation of biological macromolecules, such as the study of non-covalent interactions of proteins. In this article, the basic principles are highlighted, some of the techniques employed are described and examples of applications are provided, with particular respect being paid to the field of characterization of biomolecules.     

Painting Proteins with Covalent Labels: What's In the Picture?

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein—ligand complexes. Highlighted here are five different mass spectrometry—based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.     

质谱及相关技术在信号传导方面的研究进展

细胞信号传导是近年来生命科学研究的热点之一。有关蛋白转录后修饰 (如蛋白质磷酸化、乙酰化、糖基化等 ) ,信号肽序列测定 ,信号传导途径和多通道调节方式 ,蛋白自折叠及构象变化 ,小分子脂类信号分子等研究由于质谱技术的快速发展而取得了突破性的进展     

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.     

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.     

The structural analysis of large noncovalent oxygen binding proteins by MALLS and ESI-MS: a review on annelid hexagonal bilayer hemoglobin and crustacean hemocyanin

Understanding the function of macromolecular complexes is related to a precise knowledge of their structure. These large complexes are often fragile high molecular mass noncovalent multimeric proteins. Classical biochemical methods for determination of their native mass and subunit composition were used to resolve their quaternary structure, sometimes leading to different models. Recently, the development of mass spectrometry and multi-angle laser light scattering (MALLS) has enabled absolute determination of native masses and subunit masses. Electrospray ionization mass spectrometry (ESI-MS) was used in denaturing and native conditions to probe subunit composition and noncovalent assemblies masses up to 2.25 MDa. In a complementary way, MALLS provides mass and size estimation in various aqueous solvents. ESI-MS method can also give insights into post-translational modifications (glycosylation, disulfide bridges ). By combining native mass and subunit composition data, structural models can be proposed for large edifices such as annelid extracellular hexagonal bilayer hemoglobins (HBL Hb) and crustacean hemocyanins (Hc). Association/dissociation mechanisms, protein-protein interactions, structural diversity among species and environmental adaptations can also be addressed with these methods. With their absolute mass determination, the very high precision of spectrometry and the versatile nature of light scattering, ESI-MS and MALLS have provided a wealth of data helping to resolve parts of controversies for HBL-Hb models and opening access to new fields of investigation in structural diversity and molecular adaptation. In this review we will focus on annelid HBL-Hb and on crustacean Hc and on the original contributions of ESI-MS and MALLS in this field.     

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。     

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.     

Analysis of food proteins and peptides by mass spectrometry-based techniques

Mass spectrometry has arguably become the core technology for the characterization of food proteins and peptides. The application of mass spectrometry-based techniques for the qualitative and quantitative analysis of the complex protein mixtures contained in most food preparations is playing a decisive role in the understanding of their nature, structure, functional properties and impact on human health. The application of mass spectrometry to protein analysis has been revolutionized in the recent years by the development of soft ionization techniques such as electrospray ionization and matrix assisted laser desorption/ionization, and by the introduction of multi-stage and ‘hybrid’ analyzers able to generate de novo amino acid sequence information. The interfacing of mass spectrometry with protein databases has resulted in entirely new possibilities of protein characterization, including the high sensitivity mapping (femtomole to attomole levels) of post-translational and other chemical modifications, protein conformations and protein–protein and protein–ligand interactions, and in general for proteomic studies, building up the core platform of modern proteomic science. MS-based strategies to food and nutrition proteomics are now capable to address a wide range of analytical questions which include issues related to food quality and safety, certification and traceability of (typical) products, and to the definition of the structure/function relationship of food proteins and peptides. These different aspects are necessarily interconnected and can be effectively understood and elucidated only by use of integrated, up-to-date analytical approaches. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of food proteins are presented, with focus on issues related to their detection, identification, and quantification, relevant for their biochemical, technological and toxicological aspects.     

质谱技术在免疫分子的结构研究中的应用

质谱技术用于生物大分子的研究具有直接、简单、快速、经济等优点。近十年来 ,基质辅助激光解吸质谱 (MALDI MS)和电喷雾质谱 (ESI MS)在免疫学领域的研究中作出了重要贡献。本文着重对抗原、抗体、抗原 抗体复合物、抗原决定簇等免疫分子结构的质谱研究作一评述。大体分为四方面内容 :免疫分子的分子量、翻译后修饰、异质性、构象变化的分析 ;质谱指纹图的取得和串联质谱测序 ;抗原 抗体复合物的证明 ;B 细胞表位和T 细胞表位序列的测定。这些研究结果对于理解免疫分子的免疫功能、对于疾病的早期诊断、对于发展新药和疫苗具有重要意义     

Mass spectrometry-based structural proteomics

Structural proteomics is the application of protein chemistry and modern mass spectrometric techniques to problems such as the characterization of protein structures and assemblies and the detailed determination of protein-protein interactions. The techniques used in structural proteomics include crosslinking, photoaffinity labeling, limited proteolysis, chemical protein modification and hydrogen/deuterium exchange, all followed by mass spectrometric analysis. None of these methods alone can provide complete structural information, but a "combination" of these complementary approaches can be used to provide enough information for answering important biological questions. Structural proteomics can help to determine, for example, the detailed structure of the interfaces between proteins that may be important drug targets and the interactions between proteins and ligands. In this review, we have tried to provide a brief overview of structural proteomics methodologies, illustrated with examples from our laboratory and from the literature.     

Drift time mass spectrometric protein hydration experiments

Recent developments in electrospray ionization opened the field of mass spectrometry for macromolecules like proteins. The combination of ionization, drift time spectrometry, and mass spectrometry combines a large field of experimental techniques. In this new field it becomes possible to understand the interactions of isolated proteins with reaction partners like water. The combination of these techniques allows one to determine structural and thermodynamic properties. The experiments presented here give a first insight into the interaction of isolated protein ions with single solvent molecules. Results are presented for proteins like cytochrome C, bovine pancreatic trypsin inhibitor and lysozyme. MD-simulations on the same proteins and conditions as similar as possible to the experiments reflect the experimental results remarkable well.     

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.     

On-line coupling of hollow fiber membranes with electrospray ionization mass spectrometry for continuous affinity selection, concentration and identification of small-molecule libraries.

Combinatorial chemistry has been widely employed in the pharmaceutical industry in the effort towards drug discovery. Rapid and sensitive screening of lead candidates among library compounds has thus imposed significant analytical challenges in recent years. This work involved the development of a continuous affinity capture and concentration system, providing cost-effective and structural analysis of drug candidates in a flow-through format. The system combines the strengths of a hollow fiber dialysis membrane of ease and speed of purification and concentration with the specificity of affinity interactions in solution. The complexes between the lead compounds and the affinity binding proteins are separated from other chemical components inside a dialysis hollow fiber as the result of their differences in size. The affinity complexes are further concentrated inside a second dialysis fiber. The concentrated drug candidates are liberated from the binding proteins in a microdialysis junction and can be directly identified using electrospray ionization mass spectrometry. Two model systems, including human serum albumin-warfarin-related compounds and anti-phenobarbital antibody-barbiturates, were employed for mechanistic studies of dialysis versus dissociation kinetics and competitive selection of drug candidates according to their binding strengths.     

Analysis of protein interaction networks using mass spectrometry compatible techniques

The ability to map protein-protein interactions has grown tremendously over the last few years, making it possible to envision the mapping of whole or targeted protein interaction networks and to elucidate their temporal dynamics. The use of mass spectrometry for the study of protein complexes has proven to be an invaluable tool due to its ability to unambiguously identify proteins from a variety of biological samples. Furthermore, when affinity purification is combined with mass spectrometry analysis, the identification of multimeric protein complexes is greatly facilitated. Here, we review recent developments for the analysis of protein interaction networks by mass spectrometry and discuss the integration of different bioinformatic tools for predicting, validating, and managing interaction datasets.     

Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes

The development of “soft” ionization methods in recent years has enabled substantial progress in the mass spectrometric characterization of macromolecules, in particular important biopolymers such as proteins and nucleic acids. In contrast to the still existing limitations for the determination of molecular weights by other ionization methods such as fast atom bombardment and plasma desorption, electrospray ionization (ESI) and matrix-assisted laser desorption have provided a breakthrough to macromolecules larger than 100 kDa. Whereas these methods have been successfully applied to determine the molecular weight and primary structure of biopolymers, the recently discovered direct characterization by ESI-MS of complexes containing noncovalent interactions (“noncovalent complexes”) opens new perspectives for supramolecular chemistry and analytical biochemistry. Unlike other ionization methods ESI-MS can be performed in homogeneous solution and under nearly physiological conditions of pH, concentration, and temperature. ESI mass spectra of biopolymers, particularly proteins, exhibit series of multiply charged macromolecular ions with charge states and distributions (“charge structures”) characteristic of structural states in solution, which enable a differentiation between native and denatured tertiary structures. In the first part of this article, fundamental principles, the present knowledge about ion formation mechanism(s) of ESI-MS, the relations between tertiary structures in solution and charge structures of macro-ions in the gas phase, and experimental preconditions for the identification of noncovalent complexes are described. The hitherto successful applications to the identification of enzyme–substrate and –inhibitor complexes, supramolecular protein–and protein–nucleotide complexes, double-stranded polynucleotides, as well as synthetic self-assembled complexes demonstrate broad potential for the direct analysis of specific noncovalent interactions. The present results suggest new applications for the characterization of supramolecular structures and molecular recognition processes that previously have not been amenable to mass spectrometry; for example, the sequence-specific oligomerization of polypeptides, antigen–antibody complexes, enzyme–and receptor–ligand interactions, and the evaluation of molecular specificity in combinatorial syntheses and self-assembled systems.     

How far can we go with structural mass spectrometry of protein complexes?

Physical interactions between proteins and the formation of stable complexes form the basis of most biological functions. Therefore, a critical step toward understanding the integrated workings of the cell is to determine the structure of protein complexes, and reveal how their structural organization dictates function. Studying the three-dimensional organization of protein assemblies, however, represents a major challenge for structural biologists, due to the large size of the complexes, their heterogeneous composition, their flexibility, and their asymmetric structure. In the last decade, mass spectrometry has proven to be a valuable tool for analyzing such noncovalent complexes. Here, I illustrate the breadth of structural information that can be obtained from this approach, and the steps taken to elucidate the stoichiometry, topology, packing, dynamics, and shape of protein complexes. In addition, I illustrate the challenges that lie ahead, and the future directions toward which the field might be heading.