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.     

Native ion mobility-mass spectrometry and related methods in structural biology

Mass spectrometry-based methods have become increasingly important in structural biology — in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

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.     

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.     

Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes

Protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology. However, applying conventional structural biology approaches is challenging for transient, dynamic, or polydisperse assemblies. There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes. Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture. In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.     

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes¹. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)^2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.     

Integrating mass spectrometry of intact protein complexes into structural proteomics

MS analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other MS approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative MS approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly advancing area.     

Finding the right balance: a personal journey from individual proteins to membrane-embedded motors: based on a lecture delivered at the 36th FEBS Congress in Torino, Italy, June 2011

It is now more than 20 years since the prophetic words of John Fenn, announcing his discovery, stated that 'Electrospray spectra have been obtained for biopolymers including oligonucleotides and proteins, the latter having molecular weights up to 130 000, with as yet no evidence of an upper limit' (Fenn JB, Mann M, Kai Meng C, Fu Wong S & Whitehouse CM (1989) Science246, 64-71). Today, with the mass spectra of intact ribosomes at 2.3 MDa becoming almost routine and the first electrospray spectra of membrane-embedded motors being recorded recently, new challenges are emerging. Knowledge of the intact mass of a protein or complex is only part of the MS information available. Data from the disruption of protein complexes in solution and gas phases are leading to subunit interaction maps and architectural models. Such models are enhanced by coupling with ion mobility in which the collision cross-section of a protein complex can be defined. Linking these attributes with knowledge of subunit dynamics and the role of post-translational modifications on the stability and interactions within complexes is increasing our understanding of the factors that stabilize and convert protein complexes between different quaternary states. From our earliest experiments, studying the folding of individual proteins, through to the characterization of membrane-embedded motors, it is clear that the full potential of electrospray in structural biology has yet to be realized. The present review offers a personal view of the transition from determining the mass of an individual protein to elucidating the structure and dynamics of heterogeneous assemblies in the megadalton mass range.     

Breaking free from the crystal lattice: Structural biology in solution to study protein degraders

Structural biology offers a versatile arsenal of techniques and methods to investigate the structure and conformational dynamics of proteins and their assemblies. The growing field of targeted protein degradation centres on the premise of developing small molecules, termed degraders, to induce proximity between an E3 ligase and a protein of interest to be signalled for degradation. This new drug modality brings with it new opportunities and challenges to structural biologists. Here we discuss how several structural biology techniques, including nuclear magnetic resonance, cryo-electron microscopy, structural mass spectrometry and small angle scattering, have been explored to complement X-ray crystallography in studying degraders and their ternary complexes. Together the studies covered in this review make a case for the invaluable perspectives that integrative structural biology techniques in solution can bring to understanding ternary complexes and designing degraders.     

Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry

Well-resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2%. The mass spectral data seem to reflect known solution-phase behavior of the studied protein assembly and have therefore been directly used to probe the protein assembly topology and stability as a function of ionic strength and pH.     

A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring‐shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light‐harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.     

The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes

After more than a decade of method development, cross-linking in combination with mass spectrometry and bioinformatics is finally coming of age. This technology now provides improved opportunities for modelling by mapping structural details of functional complexes in solution. The structure of proteins or protein complexes is ascertained by identifying amino acid pairs that are positioned in close proximity to each other. The validity of this technique has recently been benchmarked for large multi-protein complexes, by comparing cross-link data with that from a crystal structure of RNA polymerase II. Here, the specific nature of this cross-linking data will be discussed to assess the technical challenges and opportunities for model building. We believe that once remaining technological challenges of cross-linking/mass spectrometry have been addressed and cross-linking/mass spectrometry data has been incorporated into modelling algorithms it will quickly become an indispensable companion of protein and protein complex modelling and a corner-stone of integrated structural biology.     

Ion mobility-mass spectrometry analysis of large protein complexes

Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.     

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.     

Subunit architecture of multimeric complexes isolated directly from cells

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.     

hu.MAP 2.0: integration of over 15,000 proteomic experiments builds a global compendium of human multiprotein assemblies

A general principle of biology is the self‐assembly of proteins into functional complexes. Characterizing their composition is, therefore, required for our understanding of cellular functions. Unfortunately, we lack knowledge of the comprehensive set of identities of protein complexes in human cells. To address this gap, we developed a machine learning framework to identify protein complexes in over 15,000 mass spectrometry experiments which resulted in the identification of nearly 7,000 physical assemblies. We show our resource, hu.MAP 2.0, is more accurate and comprehensive than previous state of the art high‐throughput protein complex resources and gives rise to many new hypotheses, including for 274 completely uncharacterized proteins. Further, we identify 253 promiscuous proteins that participate in multiple complexes pointing to possible moonlighting roles. We have made hu.MAP 2.0 easily searchable in a web interface (http://humap2.proteincomplexes.org/), which will be a valuable resource for researchers across a broad range of interests including systems biology, structural biology, and molecular explanations of disease.     

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.     

Advances in ion mobility spectrometry–mass spectrometry reveal key insights into amyloid assembly

Interfacing ion mobility spectrometry to mass spectrometry (IMS–MS) has enabled mass spectrometric analyses to extend into an extra dimension, providing unrivalled separation and structural characterization of lowly populated species in heterogeneous mixtures. One biological system that has benefitted significantly from such advances is that of amyloid formation. Using IMS–MS, progress has been made into identifying transiently populated monomeric and oligomeric species for a number of different amyloid systems and has led to an enhanced understanding of the mechanism by which small molecules modulate amyloid formation. This review highlights recent advances in this field, which have been accelerated by the commercial availability of IMS–MS instruments. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein–protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.