Multiplexed Quantitative Analysis of Antibody-Drug Conjugates with Labile CBI-Dimer Payloads In Vivo Using Immunoaffinity LC-MS/MS.

Quantitative analysis of antibody-drug conjugates (ADCs) involves cleavage of ADCs into smaller analytes representing different components and subsequent measurements from multiple assays for a more comprehensive pharmacokinetic (PK) assessment. Multiple PK analytes including the drug remaining conjugated to the antibody (or antibody-conjugated drug, acDrug) and total antibody can be accessed simultaneously using a multiplex assay by proteolytic digestion of an ADC, if the sites of conjugation are homogeneous for an ADC and the linker drug is stable to proteases. Herein, a multiplexed immunoaffinity liquid chromatography-mass spectrometry (LC-MS)/MS PK assay is described involving immunoaffinity enrichment, enzymatic conversion of prodrug, trypsin digestion, and LC-MS/MS as applied to next-generation ADCs constructed from linker drugs bearing dimeric cyclopropabenzindole (CBI) payloads (duocarmycin analogues). The cytotoxic payload is chemically labile, requiring extensive optimization in sample preparation steps to stabilize the drug without ex vivo modification and to convert the prodrug into a single active form of the drug. The qualification data for this assay format showed that this approach provides robust acDrug and total antibody data and can be extended to ADCs with different monoclonal antibody frameworks and linker chemistries. Applications of this multiplexed assay to support preclinical studies are presented.

Method development of a novel PK assay for antibody-conjugated drug measurement of ADCs using peptide-linker drug analyte.

Pharmacokinetic analysis of antibody-drug conjugates (ADCs) requires characterization and quantification of both the antibody-conjugated cytotoxic drug molecule (acDrug) as well as the antibody vehicle, among other analytes, in order to assess the safety and efficacy of ADCs. Due to the complexity of biological matrices, immunoaffinity capture is widely used for enrichment of the biotherapeutic, followed by enzymatic or chemical release of the drug and LC-MS/MS analysis to provide the concentration of acDrug. This bioanalytical strategy has been used successfully with ADCs, but is limited to ADCs having cleavable linkers. Herein, we developed a sensitive and specific method that involved subjecting the ADC to tryptic digestion, and measured a peptide that included cysteine conjugated to the drug to provide quantification of acDrug. Using this method for a THIOMAB™ antibody-drug conjugate (TDC) conjugated to MMAE via a cleavable linker, valine-citrulline, we compared peptide-linker MMAE data from the new assay format with earlier MMAE data for acDrug. This showed that the new assay format provides robust acDrug as well as total antibody concentration to study in vitro stability of the TDC in multiple matrices and in vivo pharmacokinetic models of TDC in rat and mouse. The data from the two orthogonal modes of acDrug analysis showed good agreement with each other, allowing us to successfully quantify acDrug to study the stability in vitro and the pharmacokinetic parameters in vivo. This new assay strategy allows acDrug quantification for ADCs with non-cleavable linkers where the resulting acDrug analyte is a peptide-linker drug.

Inter-domain communication of human cystathionine ß-synthase: structural basis of S-adenosyl-L-methionine activation.

Cystathionine ß-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.

Detection of conformation types of cyclosporin retaining intramolecular hydrogen bonds by mass spectrometry.

Cyclosporin is a family of neutral cyclic undecapeptides widely used for the prevention of organ transplant rejection and controlling viral infection. The equilibrium of conformations assumed by cyclosporin A in response to the solvent environment is thought to play a critical role in enabling good membrane penetration, which improves upon shielding the polarity of the molecule through forming intramolecular hydrogen bonds. However, the distribution of structures and their internal hydrogen bond geometries have not been elucidated thus far across the series of cyclosporins. Herein, we elucidate the conformational heterogeneity of cyclosporins using a set of analytical approaches including ion mobility mass spectrometry, hydrogen-deuterium exchange, and molecular dynamics simulation. Ion mobility measurements reveal a specific conformational distribution for each cyclosporin derivative in a structure-dependent manner. In general, we observe that the more compact conformer is associated with a greater frequency of intramolecular hydrogen bonds. Cyclosporin A is populated by structures with an extensive hydrogen bond network that is lacking in cyclosporin H, which is composed predominantly of a single compact conformation. The slower dynamics of cyclosporin H backbone is also consistent with the lack of hydrogen bonds. Furthermore, we find a strong correlation between the steric bulk of the side chain at position 2 of cyclosporin and the distribution of conformers due to differential accommodation of side chains within the macrocycle, and also report a wide range of conformational dynamics in solution.

Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry.

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.

Dramatically stabilizing multiprotein complex structure in the absence of bulk water using tuned Hofmeister salts.

The role that water plays in the salt-based stabilization of proteins is central to our understanding of protein biophysics. Ion hydration and the ability of ions to alter water surface tension are typically invoked, along with direct ion-protein binding, to describe Hofmeister stabilization phenomena observed for proteins experimentally, but the relative influence of these forces has been extraordinarily difficult to measure directly. Recently, we have used gas-phase measurements of proteins and large multiprotein complexes, using a combination of innovative ion mobility (IM) and mass spectrometry (MS) techniques, to assess the ability of bound cations and anions to stabilize protein ions in the absence of the solvation forces described above. Our previous work has studied a broad set of 12 anions bound to a range of proteins and protein complexes, and while primarily motivated by the analytical challenges surrounding the gas-phase measurement of solution-phase relevant protein structures, our work has also lead to a detailed physical mechanism of anion-protein complex stabilization in the absence of bulk solvent. Our more-recent work has screened a similarly-broad set of cations for their ability to stabilize gas-phase protein structure, and we have discovered surprising differences between the operative mechanisms for cations and anions in gas-phase protein stabilization. In both cases, cations and anions affect protein stabilization in the absence of solvent in a manner that is generally reversed relative to their ability to stabilize the same proteins in solution. In addition, our evidence suggests that the relative solution-phase binding affinity of the anions and cations studied here is preserved in our gas-phase measurements, allowing us to study the influence of such interactions in detail. In this report, we collect and summarize such gas-phase measurements to distill a generalized picture of salt-based protein stabilization in the absence of bulk water. Further, we communicate our most recent efforts to study the combined effects of stabilizing cations and anions on gas-phase proteins, and identify those salts that bear anion/cation pairs having the strongest stabilizing influence on protein structures

Amyloid-ß-neuropeptide interactions assessed by ion mobility-mass spectrometry.

Recently, small peptides have been shown to modulate aggregation and toxicity of the amyloid-ß protein (Aß). As such, these new scaffolds may help discover a new class of biotherapeutics useful in the treatment of Alzheimer's disease. Many of these inhibitory peptide sequences have been derived from natural sources or from Aß itself (e.g., C-terminal Aß fragments). In addition, much earlier work indicates that tachykinins, a broad class of neuropeptides, display neurotrophic properties, presumably through direct interactions with either Aß or its receptors. Based on this work, we undertook a limited screen of neuropeptides using ion mobility-mass spectrometry to search for similar such peptides with direct Aß binding properties. Our results reveal that the neuropeptides leucine enkephalin (LE) and galanin interact with both the monomeric and small oligomeric forms of Aß(1-40) to create a range of complexes having diverse stoichiometries, while some tachyknins (i.e., substance P) do not. LE interacts with Aß more strongly than galanin, and we utilized ion mobility-mass spectrometry, molecular dynamics simulations, gel electrophoresis/Western blot, and transmission electron microscopy to study the influence of this peptide on the structure of Aß monomer, small Aß oligomers, as well as the eventual formation of Aß fibrils. We find that LE binds selectively within a region of Aß between its N-terminal tail and hydrophobic core. Furthermore, our data indicate that LE modulates fibril generation, producing shorter fibrillar aggregates when added in stoichiometric excess relative to Aß.

Insights into antiamyloidogenic properties of the green tea extract (-)-epigallocatechin-3-gallate toward metal-associated amyloid-ß species.

Despite the significance of Alzheimer's disease, the link between metal-associated amyloid-ß (metal-Aß) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal-Aß 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)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]-Aß and metal-free Aß species. We found that EGCG interacted with metal-Aß species and formed small, unstructured Aß aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metal-free Aß and metal-Aß 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 Aß monomers and dimers, generating more compact peptide conformations than those from EGCG-untreated Aß species; and (ii) ternary EGCG-metal-Aß complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal-Aß species with a structure-based mechanism.

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.

Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G.

The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.

Exploring the reactivity of flavonoid compounds with metal-associated amyloid-ß species.

Metal ions associated with amyloid-ß (Aß) peptides have been suggested to be involved in the development of Alzheimer's disease (AD), but this remains unclear and controversial. Some attempts to rationally design or select small molecules with structural moieties for metal chelation and Aß interaction (i.e., bifunctionality) have been made to gain a better understanding of the hypothesis. In order to contribute to these efforts, four synthetic flavonoid derivatives FL1-FL4 were rationally selected according to the principles of bifunctionality and their abilities to chelate metal ions, interact with Aß, inhibit metal-induced Aß aggregation, scavenge radicals, and regulate the formation of reactive oxygen species (ROS) were studied using physical methods and biological assays. The compounds FL1-FL3 were able to chelate metal ions, but showed limited solubility in aqueous buffered solutions. In the case of FL4, which was most compatible with aqueous conditions, its binding affinities for Cu(2+) and Zn(2+) (nM and µM, respectively) were obtained through solution speciation studies. The direct interaction between FL4 and Aß monomer was weak, which was monitored by NMR spectroscopy and mass spectrometry. Employing FL1-FL4, no noticeable inhibitory effect on metal-mediated Aß aggregation was observed. Among FL1-FL4, FL3, having 3-OH, 4-oxo, and 4'-N(CH(3))(2) groups, exhibited similar antioxidant activity to the vitamin E analogue, Trolox, and ca. 60% reduction in the amount of hydrogen peroxide (H(2)O(2)) generated by Cu(2+)-Aß in the presence of dioxygen (O(2)) and a reducing agent. Overall, the studies here suggest that although four flavonoid molecules were selected based on expected bifunctionality, their properties and metal-Aß reactivity were varied depending on the structure differences, demonstrating that bifunctionality must be well tuned to afford desirable reactivity.

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.

Ion mobility-mass spectrometry reveals conformational changes in charge reduced multiprotein complexes.

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, 'native-like' geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.

Bound anions differentially stabilize multiprotein complexes in the absence of bulk solvent.

The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics, and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility-mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting 'shell' of counterions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility-mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of 12 different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counterions on gas-phase protein structure and allows us to rank and classify counterions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride, and nitrate anions are among the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion-protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counterion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent.

A two-site mechanism for the inhibition of IAPP amyloidogenesis by zinc.

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting ß-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.

Characterizing the resolution and accuracy of a second-generation traveling-wave ion mobility separator for biomolecular ions.

High-accuracy, high-resolution ion mobility measurements enable a vast array of important contemporary applications in biological chemistry. With the recent advent of both new, widely available commercial instrumentation and also new calibration datasets tailored for the aforementioned commercial instrumentation, the possibilities for extending such high performance measurements to a diverse set of applications have never been greater. Here, we assess the performance characteristics of a second-generation traveling-wave ion mobility separator, focusing on those figures of merit that lead to making measurements of collision cross-section having both high precision and high accuracy. Through performing a comprehensive survey of instrument parameters and settings, we find instrument conditions for optimized drift time resolution, cross-section resolution, and cross-section accuracy for a range of peptide, protein and multi-protein complex ions. Moreover, the conditions for high accuracy IM results are significantly different from those optimized for separation resolution, indicating that a balance between these two metrics must be attained for traveling wave IM separations of biomolecules. We also assess the effect of ion heating during IM separation on instrument performance.

Retinol and retinol-binding protein stabilize transthyretin via formation of retinol transport complex.

Transthyretin (TTR) is a plasma hormone carrier protein associated with hereditary and senile forms of systemic amyloid disease, wherein slow tetramer disassembly is thought to be an obligatory step. Plasma transport of retinol is carried out exclusively by the retinol-binding protein (RBP), through complexation with transthyretin. Using mass spectrometry to examine the subunit exchange dynamics, we find that retinol stabilizes the quaternary structure of transthyretin, through its interactions with RBP, reducing the rate of transthyretin disassembly ∼17-fold compared to apoTTR. In the absence of retinol but in the presence of RBP, transthyretin is only marginally stabilized with the rate of disassembly reduced ∼two-fold with respect to apoTTR. Surprisingly, we found two retinoids that stabilize transthyretin directly, in the absence of RBP, whereas retinol itself requires RBP in order to stabilize transthyretin. Our results demonstrate new roles for RBP and retinoids as stabilizers of transthyretin.

Integrating ion mobility mass spectrometry with molecular modelling to determine the architecture of multiprotein complexes.

Current challenges in the field of structural genomics point to the need for new tools and technologies for obtaining structures of macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) and incomplete atomic structures, usually from X-ray crystallography, to generate models of the subunit architecture of protein complexes. We begin by analyzing protein complexes using IM-MS, and by taking measurements of both intact complexes and sub-complexes that are generated in solution. We then examine available high resolution structural data and use a suite of computational methods to account for missing residues at the subunit and/or domain level. High-order complexes and sub-complexes are then constructed that conform to distance and connectivity constraints imposed by IM-MS data. We illustrate our method by applying it to multimeric protein complexes within the Escherichia coli replisome: the sliding clamp, (beta2), the gamma complex (gamma3deltadelta'), the DnaB helicase (DnaB6) and the Single-Stranded Binding Protein (SSB4).

Alternate dissociation pathways identified in charge-reduced protein complex ions.

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.