Search

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1β. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM-MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1β, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1β monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by ∼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis.

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.

Membrane proteins represent most current therapeutic targets, yet remain understudied due to their insolubility in aqueous solvents and generally low yields during purification and expression. Ion mobility-mass spectrometry and collision induced unfolding experiments have recently garnered attention as methods capable of directly detecting and quantifying ligand binding within a wide range of membrane protein systems. Despite prior success, ionized surfactant often creates chemical noise patterns resulting in significant challenges surrounding the study of small membrane protein-ligand complexes. Here, we present a new data analysis workflow that overcomes such chemical noise and then utilize this approach to quantify and classify ligand binding associated with the 36 kDa dimer of translocator protein (TSPO). Following our denoising protocol, we detect separate gas-phase unfolding signatures for lipid and protoporphyrin TSPO binders, molecular classes that likely interact with separate regions of the protein surface. Further, a detailed classification analysis reveals that lipid alkyl chain saturation levels can be detected within our gas-phase protein unfolding data. We combine these data and classification schemes with mass spectra acquired directly from liquid-liquid extracts to propose an identity for a previously unknown endogenous TSPO ligand.

Collision-induced unfolding (CIU) of protein ions and their noncovalent complexes offers relatively rapid access to a rich portfolio of biophysical information, without the need to tag or purify proteins prior to analysis. Such assays have been characterized extensively for a range of therapeutic proteins, proving exquisitely sensitive to alterations in protein sequence, structure, and post-translational modification state. Despite advantages over traditional probes of protein stability, improving the throughput and information content of gas-phase protein unfolding assays remains a challenge for current instrument platforms. In this report, we describe modifications to an Agilent 6560 drift tube ion mobility-mass spectrometer in order to perform robust, simultaneous CIU across all precursor ions detected. This approach dramatically increases the speed associated with typical CIU assays, which typically involve mass selection of narrow m/ z regions prior to collisional activation, and thus their development requires a comprehensive assessment of charge-stripping reactions that can unintentionally pollute CIU data with chemical noise when more than one precursor ion is allowed to undergo simultaneous activation. By studying the unfolding and dissociation of intact antibody ions, a key analyte class associated with biotherapeutics, we reveal a predictive relationship between the precursor charge state, the amount of buffer components bound to the ions of interest, and the amount of charge stripping detected. We then utilize our knowledge of antibody charge stripping to rapidly capture CIU data for a range of antibody subclasses and subtypes across all charge states simultaneously, demonstrating a strong charge state dependence on the information content of CIU. Finally, we demonstrate that CIU data collection times can be further reduced by scanning fewer voltage steps, enabling us to optimize the throughput of our improved CIU methods and confidently differentiate antibody variant ions using ∼20% of the data typically collected during CIU. Taken together, our results characterize a new instrument platform for biotherapeutic stability measurements with dramatically improved throughput and information content.

The combination of ion-mobility (IM) separation with mass spectrometry (MS) has impacted global measurement efforts in areas ranging from food analysis to drug discovery. Reasons for the broad adoption of IM-MS include its significantly increased peak capacity, duty-cycle, and ability to reconstruct fragmentation data in parallel, all of which greatly enable the analyses of complex mixtures. More fundamentally, however, measurements of ion-gas molecule collision cross sections (CCSs) are used to support compound identification and quantitation efforts as well as study the structures of large biomolecules. As the first commercialized form of IM-MS, Traveling Wave Ion Mobility (TWIM) devices are operated at low pressures (∼3 mbar) and voltages, are relatively short (∼25 cm), and separate ions on a timescale of tens of milliseconds. These qualities make TWIM ideally suited for hybridization with MS. Owing to the complicated motion of ions in TWIM devices, however, IM transit times must be calibrated to enable CCS measurements. Applicability of these calibrations has hitherto been restricted to primarily singly charged small molecules and some classes of large, multiply charged ions under a significantly narrower range of instrument conditions. Here, we introduce and extensively characterize a dramatically improved TWIM calibration methodology. Using over 2500 experimental TWIM data sets, covering ions that span over 3.5 orders of magnitude of molecular mass, we demonstrate robust calibrations for a significantly expanded range of instrument conditions, thereby opening up new analytical application areas and enabling the expansion of high-precision CCS measurements for both existing and next-generation TWIM instrumentation.

Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb), Inflectra/Remsima (Celltrion), based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility, and quantitative peptide mapping. The levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact. Importantly, we identified more than 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13.2% of Remsima glycoforms, which translated into a 2-fold reduction in the level of FcγIIIa receptor binding for Remsima. While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely because of methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

Analysis of protein complexes by ion mobility-mass spectrometry is a valuable method for the rapid assessment of complex composition, binding stoichiometries, and structures. However, capturing labile, unknown protein assemblies directly from cells remains a challenge for the technology. Furthermore, ion mobility-mass spectrometry measurements of complexes, subcomplexes, and subunits are necessary to build complete models of intact assemblies, and such data can be difficult to acquire in a comprehensive fashion. Here, we present the use of novel mass spectrometry cleavable cross-linkers and tags to stabilize intact protein complexes for ion mobility-mass spectrometry. Our data reveal that tags and linkers bearing permanent charges are superior stabilizers relative to neutral cross-linkers, especially in the context of retaining compact forms of the assembly under a wide array of activating conditions. In addition, when cross-linked protein complexes are collisionally activated in the gas phase, a larger proportion of the product ions produced are often more compact and reflect native protein subcomplexes when compared with unmodified complexes activated in the same fashion, greatly enabling applications in structural biology.

Top-down proteomics has emerged as a transformative method for the analysis of protein sequence and post-translational modifications (PTMs). Top-down experiments have historically been performed primarily on ultrahigh resolution mass spectrometers due to the complexity of spectra resulting from fragmentation of intact proteins, but recent advances in coupling ion mobility separations to faster, lower resolution mass analyzers now offer a viable alternative. However, software capable of interpreting the highly complex two-dimensional spectra that result from coupling ion mobility separation to top-down experiments is currently lacking. In this manuscript we present a software suite consisting of two programs, IMTBX ("IM Toolbox") and Grppr ("Grouper"), that enable fully automated processing of such data. We demonstrate the capabilities of this software suite by examining a series of intact proteins on a Waters Synapt G2 ion-mobility equipped mass spectrometer and compare the results to the manual and semiautomated data analysis procedures we have used previously.

Electrospray ionization coupled to mass spectrometry is a key technology for determining the stoichiometries of multiprotein complexes. Despite highly accurate results for many assemblies, challenging samples can generate signals for artifact protein-protein binding born of the crowding forces present within drying electrospray droplets. Here, for the first time, we study the formation of preferred protein quaternary structures within such rapidly evaporating nanodroplets. We use ion mobility and tandem mass spectrometry to investigate glutamate dehydrogenase dodecamers and serum amyloid P decamers as a function of protein concentration, along with control experiments using carefully chosen protein analogues, to both establish the formation of operative mechanisms and assign the bimodal conformer populations observed. Further, we identify an unprecedented symmetric collision-induced dissociation pathway that we link directly to the quaternary structures of the precursor ions selected.

Multiprotein complexes have three-dimensional shapes and dynamic functions that impact almost every aspect of biochemistry. Despite this, our ability to rapidly assess the structures of such macromolecules lags significantly behind high-throughput efforts to identify their function, especially in the context of human disease. Here, we describe results obtained by coupling ion mobility-mass spectrometry with automated robotic sampling of different solvent compositions. This combination of technologies has allowed us to explore an extensive set of solution conditions for a group of eight protein homotetramers, representing a broad sample of protein structure and stability space. We find that altering solution ionic strength in concert with dimethylsulfoxide content is sufficient to disrupt the protein-protein interfaces of all of the complexes studied here. Ion mobility measurements captured for both intact assemblies and subcomplexes match expected values from available X-ray structures in all cases save two. For these exceptions, we find that distorted subcomplexes result from extreme disruption conditions, and are accompanied by small shifts in intact tetramers size, thus enabling the removal of distorted subcomplex data in downstream models. Furthermore, we find strong correlations between the relative intensities of disrupted protein tetramers and the relative number and type of interactions present at interfaces as a function of disrupting agent added. In most cases, this correlation appears strong enough to quantify various types of protein interfacial interactions within unknown proteins following appropriate calibration.

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.

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.

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-ion mobility-orthogonal TOF mass spectrometer is demonstrated. SID of approximately 100-fmol amounts of model peptides HLGLAR (m/z 666.8), gramicidin S (m/z 1142.5), and bovine insulin b chain (m/z 3495.5) was accomplished using hydrocarbon-coated gold grids and approximately 20-eV collision energies. The current version of the instrument achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (approximately 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOF-MS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (e.g., peptide mass mapping) and partial sequence analysis, (e.g., real-time tandem mass spectrometry.)

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.

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.

An ion mobility-mass spectrometry technique for rapid screening of phosphopeptides in protein digests is described. A data set of 43 sequences (ranging in mass from 400 to 3000 m/z) of model and tryptic peptides, including serine, threonine, and tyrosine phosphorylation, was investigated, and the data support our previously reported observation (Ruotolo, B. T.; Verbeck, G. F., IV; Thomson, L. M.; Woods, A. S.; Gillig, K. J.; Russell, D. H. J. Proteome Res. 2002, 1, 303.) that the drift time-m/z relationship for singly charged phosphorylated peptide ions is different from that for nonphosphorylated peptides. The data further illustrate that a combined data-dependent IM-MS/MS approach for phosphopeptide screening would have enhanced throughput over conventional MS/MS-based methodologies.