Recent advancements place a comprehensive catalog of protein structure, oligomeric state, sequence, and modification status tentatively within reach, thus providing an unprecedented roadmap to ...therapies for many human diseases. To achieve this goal, revolutionary technologies capable of bridging key gaps in our ability to simultaneously measure protein composition and structure must be developed. Much of the current progress in this area has been catalyzed by mass spectrometry (MS) tools, which have become an indispensable resource for interrogating the structural proteome. For example, methods associated with native proteomics seek to comprehensively capture and quantify the endogenous assembly states for all proteins within an organism. Such technologies have often been partnered with ion mobility (IM) separation, from which collision cross section (CCS) information can be rapidly extracted to provide protein size information. IM technologies are also being developed that utilize CCS values to enhance the confidence of protein identification workflows derived from liquid chromatography-IM-MS analyses of enzymatically produced peptide mixtures. Such parallel advancements in technology beg the question: can CCS values prove similarly useful for the identification of intact proteins and their complexes in native proteomics? In this perspective, I examine current evidence and technology trends to explore the promise and limitations of such CCS information for the comprehensive analysis of multiprotein complexes from cellular mixtures.
The three‐dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling ...the generation of models for both proteins and their higher‐order assemblies. While important coursed‐grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas‐phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility‐mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. This correlation and its potential uses for structural biology is discussed.
Gas‐phase unfolding is used as a means to determine the number of autonomously folded domains within monomeric proteins. Ion‐mobility mass spectrometry data show a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. CCS=collision cross‐section.
Monoclonal antibodies (mAbs) are among the fastest growing class of therapeutics due to their high specificity and low incidence of side effects. Unlike most drugs, mAbs are complex macromolecules ...(∼150 kDa), leading to a host of quality control and characterization challenges inherent in their development. Recently, we introduced a new approach for the analysis of the intact proteins based on ion mobility-mass spectrometry (IM-MS). Our protocol involves the collision induced unfolding (CIU) of intact antibodies, where collisional heating in the gas-phase is used to generate unfolded antibody forms, which are subsequently separated by IM and then analyzed by MS. Collisional energy is added to the antibody ions in a stepwise fashion, and “fingerprint plots” are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. In this report, we have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonding and general levels of glycosylation. In addition, we validate our CIU protocols through control experiments and systematic statistical evaluations of CIU reproducibility. We conclude by projecting the impact of our approach for antibody-related drug discovery and development applications.
•Collision induced unfolding (CIU) uses gas phase activation to probe biomolecules.•CIU can detect subtle changes in protein structure, stability or composition.•CIU can probe a wide range of ...biomolecules, from multiprotein complexes to biotherapeutics.•CIU has potential as a high throughput technique, differentiating states in seconds.
Rapidly characterizing the three-dimensional structures of proteins and the multimeric machines they form remains one of the great challenges facing modern biological and medical sciences. Ion mobility–mass spectrometry based techniques are playing an expanding role in characterizing these functional complexes, especially in drug discovery and development workflows. Despite this expansion, ion mobility–mass spectrometry faces many challenges, especially in the context of detecting small differences in protein tertiary structure that bear functional consequences. Collision induced unfolding is an ion mobility–mass spectrometry method that enables the rapid differentiation of subtly-different protein isoforms based on their unfolding patterns and stabilities. In this review, we summarize the modern implementation of such gas-phase unfolding experiments and provide an overview of recent developments in both methods and applications.
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•Collision induced unfolding (CIU) can detect differences in the glycostructures of intact antibody ions.•CIU comparison plots reveal that both the ground state structures and ...stabilities of intact antibodies are influenced by altered glycosylation.•A greater sensitivity to changes in antibody glycosylation is achieved when the Fc fragment is released and analyzed.
Glycosylation has a significant impact on the effector function, immunogenicity, plasmatic clearance, and resistance towards proteases for monoclonal antibodies. Antibody glycoforms directly result in a form of structural heterogeneity due to the variety of sugar moieties and the available range of assembly states for the sugar linkages involved. Thus, the rapid characterization of antibody glycosylation is a critical yet challenging objective in the development of antibody-based therapeutics. Recently, we introduced an ion mobility-mass spectrometry approach for intact antibody analysis, which involves the collision induced unfolding of intact antibody ions. In this report, we demonstrate the use of such gas-phase unfolding analyses to differentiate subtly-different glycoforms within both intact antibody monomers and antibody Fc fragments, using minimal sample preparation and purification. We find evidence for a strong correlation between the gas-phase stabilities of antibody ions and the number of sugars attached to their sequences. We conclude by projecting the utility of our gas-phase unfolding assay in the context of antibody characterization workflows aimed at differentiating antibody glycoforms that cannot be readily resolved by MS alone.
The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to ...modern measurement science. Mass spectrometry has evolved into a rapid and sensitive tool for assessing the structures, stabilities, and dynamics of such proteins. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics. We conclude by discussing the future of structural mass spectrometry, and its role in enabling the biopharmaceutical pipeline.
The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to modern measurement science. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics.
► Mass spectrometry is a sensitive, high-resolution means for determining the oligomeric distribution of proteins. ► The different oligomers and conformers comprising a heterogeneous ensemble of ...proteins can be individually interrogated. ► Intra- and inter-subunit connectivity, solvent accessibility, and oligomeric size can be elucidated. ► Pre-equilibrium and equilibrium dynamics spanning residue to oligomer levels can be measured on the μs to hour timescales. ► Mass spectrometry is an approach of wide applicability which can be integrated into hybrid structural biology approaches.
Over the past two decades, mass spectrometry (MS) has emerged as a
bone fide approach for structural biology. MS can inform on all levels of protein organization, and enables quantitative assessments of their intrinsic dynamics. The key advantages of MS are that it is a sensitive, high-resolution separation technique with wide applicability, and thereby allows the interrogation of transient protein assemblies in the context of complex mixtures. Here we describe how molecular-level information is derived from MS experiments, and how it can be combined with spatial and dynamical restraints obtained from other structural biology approaches to allow hybrid studies of protein architecture and movements.
Ion mobility–mass spectrometry (IM–MS) has become an important addition to the structural biology toolbox, but separating closely related protein conformations remain challenging. Collision-induced ...unfolding (CIU) has emerged as a valuable technique for distinguishing iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. The speed and sensitivity of CIU analyses, coupled with their information-rich data sets, have resulted in the rapid growth of CIU for applications, ranging from the structural assessment of protein complexes to the characterization of biotherapeutics. This growth has occurred despite a lag in the capabilities of informatics tools available to process the complex data sets generated by CIU experiments, resulting in laborious manual analysis remaining commonplace. Here, we present CIUSuite 2, a software suite designed to enable robust, automated analysis of CIU data across the complete range of current CIU applications and to support the implementation of CIU as a true high-throughput technique. CIUSuite 2 uses statistical fitting and modeling methods to reliably quantify features of interest within CIU data sets, particularly in data with poor signal quality that cannot be interpreted with existing analysis tools. By reducing the signal-to-noise requirements for handling CIU data, we are able to demonstrate reductions in acquisition time of up to 2 orders of magnitude over current workflows. CIUSuite 2 also provides the first automated system for classifying CIU fingerprints, enabling the next generation of ligand screening and structural analysis experiments to be accomplished in a high-throughput fashion.
Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass ...spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm−3, which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.
Despite the significance of Alzheimer's disease, the link between metal-associated amyloid-β (metal-Aβ) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of ...specifically targeting and modulating metal-Aβ species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (-)-epigallocatechin-3-gallate (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4HJihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG, with metal Cu(II) and Zn(II)-Aβ and metal-free Aβ species. We found that EGCG interacted with metal-Aβ species and formed small, unstructured Aβ aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metalfree Aβ and metal-Aβ was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aβ monomers and dinners, generating more compact peptide conformations than those from EGCGuntreated Aβ species; and (ii) ternary EGCG-metal-Aβ complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal-Aβ species with a structurebased mechanism.
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