Cedric E. Bobst

H/D Exchange and Mass Spectrometry in the Studies of Protein Conformation and Dynamics: Is There a Need for a Top-Down Approach?

Hydrogen/deuterium exchange (HDX) combined with mass spectrometry (MS) detection has matured in recent years to become a powerful tool in structural biology and biophysics. Several limitations of this technique can and will be addressed by tapping into the ever expanding arsenal of methods to manipulate ions in the gas phase offered by mass spectrometry.

Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics.

Biopharmaceuticals are a unique class of medicines due to their extreme structural complexity. The structure of these therapeutic proteins is critically important for their efficacy and safety, and the ability to characterize it at various levels (from sequence to conformation) is critical not only at the quality control stage, but also throughout the discovery and design stages. Biological mass spectrometry (MS) offers a variety of approaches to study structure and behavior of complex protein drugs and has already become a default tool for characterizing the covalent structure of protein therapeutics, including sequence and post-translational modifications. Recently, MS-based methods have also begun enjoying a dramatic growth in popularity as a means to provide information on higher order structure and dynamics of biotechnology products. In particular, hydrogen/deuterium exchange MS and charge state distribution analysis of protein ions in electrospray ionization (ESI) MS offer a convenient way to assess the integrity of protein conformation. Native ESI MS also allows the interactions of protein drugs with their therapeutic targets and other physiological partners to be monitored using simple model systems. MS-based methods are also applied to study pharmacokinetics of biopharmaceutical products, where they begin to rival traditional immunoassays. MS already provides valuable support to all stages of development of biopharmaceuticals, from discovery to post-approval monitoring, and its impact on the field of biopharmaceutical analysis will undoubtedly continue to grow.

Conformation and dynamics of biopharmaceuticals: Transition of mass spectrometry-based tools from academe to industry

Mass spectrometry plays a very visible role in biopharmaceutical industry, although its use in development, characterization, and quality control of protein drugs is mostly limited to the analysis of covalent structure (amino acid sequence and post-translational modifications). Despite the centrality of protein conformation to biological activity, stability, and safety of biopharmaceutical products, the expanding arsenal of mass spectrometry-based methods that are currently available to probe higher order structure and conformational dynamics of biopolymers did not, until recently, enjoy much attention in the industry. This is beginning to change as a result of recent work demonstrating the utility of these experimental tools for various aspects of biopharmaceutical product development and manufacturing. In this work, we use a paradigmatic protein drug interferon β-1a as an example to illustrate the utility of mass spectrometry as a powerful tool not only to assess the integrity of higher order structure of a protein drug, but also to predict consequences of its degradation at a variety of levels.

Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

Mass spectrometry‐based methods to study protein architecture and dynamics

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post‐translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini‐review highlights several popular mass spectrometry‐based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry.

Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry

Significance Structure and dynamic features of nonnative protein conformations are critically important for a variety of processes, such as folding, recognition, binding, aggregation, and enzyme catalysis to name just a few. Nevertheless, detailed structural characterization of these elusive species is difficult, because they almost always coexist in equilibrium with other conformers and cannot be isolated prior to analysis. As a result, most studies report structural features that are averaged across the entire protein ensemble, rather than unique features of individual conformers. This paper reports structural characterization of an individual nonnative protein conformer without interference from other states coexisting in solution under equilibrium. Characterization of structure and dynamics of nonnative protein states is important for understanding molecular mechanisms of processes as diverse as folding, binding, aggregation, and enzyme catalysis to name just a few; however, selectively probing local minima within rugged energy landscapes remains a problem. Mass spectrometry (MS) coupled with hydrogen/deuterium exchange (HDX) offers a unique advantage of being able to make a distinction among multiple protein conformers that coexist in solution; however, detailed structural interrogation of such states previously remained out of reach of HDX MS. In this work, we exploited the aforementioned unique feature of HDX MS in combination with the ability of MS to isolate narrow populations of protein ions to characterize individual protein conformers coexisting in solution in equilibrium. Subsequent fragmentation of the protein ions using electron-capture dissociation allowed us to allocate the deuterium distribution along the protein backbone, yielding a backbone-amide protection map for the selected conformer unaffected by contributions from other protein states present in solution. The method was tested with the small regulatory protein ubiquitin (Ub), which is known to form nonnative intermediate states under a variety of mildly denaturing conditions. Protection maps of these intermediate states obtained at residue-level resolution provide clear evidence that they are very similar to the so-called A-state of Ub that is formed in solutions with low pH and high alcohol. Method validation was carried out by comparing the backbone-amide protection map of native Ub with those deduced from high-resolution NMR measurements.

Impact of oxidation on protein therapeutics: Conformational dynamics of intact and oxidized acid‐β‐glucocerebrosidase at near‐physiological pH

The solution dynamics of an enzyme acid‐β‐glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX‐MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40–60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking.

Advanced mass spectrometry-based methods for the analysis of conformational integrity of biopharmaceutical products.

Mass spectrometry has already become an indispensable tool in the analytical armamentarium of the biopharmaceutical industry, although its current uses are limited to characterization of covalent structure of recombinant protein drugs. However, the scope of applications of mass spectrometry-based methods is beginning to expand to include characterization of the higher order structure and dynamics of biopharmaceutical products, a development which is catalyzed by the recent progress in mass spectrometry-based methods to study higher order protein structure. The two particularly promising methods that are likely to have the most significant and lasting impact in many areas of biopharmaceutical analysis, direct ESI MS and hydrogen/deuterium exchange, are focus of this article.

A new approach to measuring protein backbone protection with high spatial resolution using H/D exchange and electron capture dissociation

Inadequate spatial resolution remains one of the most serious limitations of hydrogen/deuterium exchange-mass spectrometry (HDX-MS), especially when applied to larger proteins (over 30 kDa). Supplementing proteolytic fragmentation of the protein in solution with ion dissociation in the gas phase has been used successfully by several groups to obtain near-residue level resolution. However, the restrictions imposed by the LC-MS/MS mode of operation on the data acquisition time frame makes it difficult in many cases to obtain a signal-to-noise ratio adequate for reliable assignment of the backbone amide protection levels at individual residues. This restriction is lifted in the present work by eliminating the LC separation step from the workflow and taking advantage of the high resolving power and dynamic range of a Fourier transform ion cyclotron resonance-mass spectrometer (FTICR-MS). A residue-level resolution is demonstrated for a peptic fragment of a 37 kDa recombinant protein (N-lobe of human serum transferrin), using electron-capture dissociation as an ion fragmentation tool. The absence of hydrogen scrambling in the gas phase prior to ion dissociation is verified using redundant HDX-MS data generated by FTICR-MS. The backbone protection pattern generated by direct HDX-MS/MS is in excellent agreement with the known crystal structure of the protein but also provides information on conformational dynamics, which is not available from the static X-ray structure.

Transferrin as a model system for method development to study structure, dynamics and interactions of metalloproteins using mass spectrometry

BACKGROUND Transferrin (Tf) is a paradigmatic metalloprotein, which has been extensively studied in the past and still is a focal point of numerous investigation efforts owing to its unique role in iron homeostasis and enormous promise as a component of a wide range of therapies. SCOPE OF REVIEW Electrospray ionization mass spectrometry (ESI MS) is a potent analytical tool that has been used successfully to study various properties of Tf and Tf-based products, ranging from covalent structure and metal binding to conformation and interaction with their physiological partners. MAJOR CONCLUSIONS Various ESI MS-based techniques produce unique information on Tf properties and behavior that is highly complementary to information provided by other experimental techniques. GENERAL SIGNIFICANCE The experimental ESI MS-based techniques developed for Tf studies are not only useful for understanding of fundamental aspects of the iron-binding properties of this protein and optimizing Tf-based therapeutic products, but can also be applied to study a range of other metalloproteins. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.