Mass spectrometry-based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact-mass level.

The intact-mass MS measurements are becoming increasingly popular in characterization of a range of biopolymers, especially those of interest to biopharmaceutical industry. However, as the complexity of protein therapeutics and other macromolecular medicines increases, the new challenges arise, one of which is the high levels of structural heterogeneity that are frequently exhibited by such products. The very notion of the molecular mass measurement loses its clear and intuitive meaning when applied to an extremely heterogenous system that cannot be characterized by a unique mass, but instead requires that a mass distribution be considered. Furthermore, convoluted mass distributions frequently give rise to unresolved ionic signal in mass spectra, from which little-to-none meaningful information can be extracted using standard approaches that work well for homogeneous systems. However, a range of technological advances made in the last decade, such as the hyphenation of intact-mass MS measurements with front-end separations, better integration of ion mobility in MS workflows, development of an impressive arsenal of gas-phase ion chemistry tools to supplement MS methods, as well as the revival of the charge detection MS and its triumphant entry into the field of bioanalysis already made impressive contributions towards addressing the structural heterogeneity challenge. An overview of these techniques is accompanied by critical analysis of the strengths and weaknesses of different approaches, and a brief overview of their applications to specific classes of biopharmaceutical products, vaccines, and nonbiological complex drugs.

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis.

Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

Characterization of Large Immune Complexes with Size Exclusion Chromatography and Native Mass Spectrometry Supplemented with Gas Phase Ion Chemistry.

Large immune complexes formed by the cross-linking of antibodies with polyvalent antigens play critical roles in modulating cell-mediated immunity. While both the size and the shape of immune complexes are important determinants in Fc receptor-mediated signaling responsible for phagocytosis, degranulation, and, in some instances, autoimmune pathologies, their characterization remains extremely challenging due to their large size and structural heterogeneity. We use native mass spectrometry (MS) supplemented with limited charge reduction in the gas phase to determine the stoichiometry of immune complexes formed by a bivalent (homodimeric) antigen, a 163 kDa aminopeptidase P2 (APP2), and a monoclonal antibody (mAb) to APP2. The observed (APP2·mAb)n complexes populate a wide range of stoichiometries (n = 1-4) with the largest detected species exceeding 1 MDa, although the gas-phase dissociation products are also evident in the mass spectra. While frequently considering a nuisance that complicates interpretation of native MS data, limited dissociation provides an additional dimension for characterization of the immune complex quaternary structure. APP2/mAb associations with identical composition but slightly different elution times in size exclusion chromatography exhibit notable differences in their spontaneous fragmentation profiles. The latter indicates the presence of both extended linear and cyclized (APP2·mAb)n configurations. The unique ability of MS to distinguish between such isomeric structures will be invaluable for a variety of applications where the biological effects of immune complexes are determined by their ability to assemble Fc receptor clusters of certain density on cell surfaces, such as platelet activation by clustering the low-affinity receptors FcγRIIa on their surface.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Rapid Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches and Multivariate Analysis.

Intact-mass measurements are becoming increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS is an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein but also the mass range populated by the entire complement of proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation (with the glycan load of phenotype 2-1 being notably lighter) despite a significant overlap of their ionic signals. More detailed characterization of their glycosylation patterns is enabled by the recently introduced technique of cross-path reactive chromatography (XP-RC) with online MS detection, which combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains and provides a wealth of information on glycosylation patterns of the heavy chains. N-Glycosylation patterns of both haptoglobin phenotypes were found to be consistent with bi- and triantennary structures of complex type that exhibit significant level of fucosylation and sialylation. However, multivariate analysis of haptoglobin 1-1 reveals higher number of the triantennary structures, in comparison to haptoglobin 2-1, as well as a higher extent of fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of the intact-mass analysis supplemented by limited charge reduction, suggesting that the latter technique can be employed in situations when fast assessment of protein heterogeneity is needed (e.g., process analytical technology applications).

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Charge Manipulation Using Solution and Gas-Phase Chemistry to Facilitate Analysis of Highly Heterogeneous Protein Complexes in Native Mass Spectrometry.

Structural heterogeneity is a significant challenge complicating (and in some cases making impossible) electrospray ionization mass spectrometry (ESI MS) analysis of noncovalent complexes comprising structurally heterogeneous biopolymers. The broad mass distribution exhibited by such species inevitably gives rise to overlapping ionic signals representing different charge states, resulting in a continuum spectrum with no discernible features that can be used to assign ionic charges and calculate their masses. This problem can be circumvented by using limited charge reduction, which utilizes gas-phase chemistry to induce charge-transfer reactions within ionic populations selected within narrow m/z windows, thereby producing well-defined and readily interpretable charge ladders. However, the ionic signal in native MS typically populates high m/z regions of mass spectra, which frequently extend beyond the precursor ion isolation limits of most commercial mass spectrometers. While the ionic signal of single-chain proteins can be shifted to lower m/z regions simply by switching to a denaturing solvent, this approach cannot be applied to noncovalent assemblies due to their inherent instability under denaturing conditions. An alternative approach explored in this work relies on adding supercharging reagents to protein solutions as a means of increasing the extent of multiple charging of noncovalent complexes in ESI MS without compromising their integrity. This shifts the ionic signal down the m/z scale to the region where ion selection and isolation can be readily accomplished with a front-end quadrupole, followed by limited charge reduction of the isolated ionic population. The feasibility of the new approach is demonstrated using noncovalent complexes formed by hemoglobin with structurally heterogeneous haptoglobin.

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor.

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community-the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native MS as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work, we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

Towards better understanding of the heparin role in NETosis: feasibility of using native mass spectrometry to monitor interactions of neutrophil elastase with heparin oligomers.

Neutrophil elastase is a serine protease released by neutrophils, and its dysregulation has been associated with a variety of debilitating pathologies, most notably cystic fibrosis. This protein is also a prominent component of the so-called neutrophil extracellular traps (NETs), whose formation is a part of the innate immunity response to invading pathogens, but also contributes to a variety of pathologies ranging from autoimmune disorders and inflammation to cancer to thrombotic complications in COVID-19. Retention of neutrophil elastase within NETs is provided by ejected DNA chains, although this protein is also capable of interacting with a range of other endogenous polyanions, such as heparin and heparan sulfate. In this work, we evaluate the feasibility of using native mass spectrometry (MS) as a means of studying interactions of neutrophil elastase with heparin oligomers ranging from structurally homogeneous synthetic pentasaccharide fondaparinux to relatively long (up to twenty saccharide units) and structurally heterogeneous chains produced by partial depolymerization of heparin. The presence of heterogeneous glycan chains on neutrophil elastase and the structural heterogeneity of heparin oligomers render the use of standard MS to study their complexes impractical. However, supplementing MS with limited charge reduction in the gas phase allows meaningful data to be extracted from MS measurements. In contrast to earlier molecular modeling studies where a single heparin-binding site was identified, our work reveals the existence of multiple binding sites, with a single protein molecule being able to accommodate up to three decasaccharides. The measurements also reveal the ability of even relatively short heparin oligomers to bridge two protein molecules, suggesting that characterization of these complexes using native MS can shed light on the structural properties of NETs. Lastly, the use of MS allows the binding preferences of heparin oligomers to neutrophil elastase to be studied with respect to specific structural properties of heparin, such as the level of sulfation (i.e., charge density). All experimental measurements are carried out in parallel with molecular dynamics simulations of the protein/heparin oligomer systems, which are in remarkable agreement with the experimental data and highlight the role of electrostatic interactions as dominant forces governing the formation of these complexes.

Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and Assembly.

Association of platelet factor 4 (PF4) with heparin is a first step in formation of aggregates implicated in the development of heparin-induced thrombocytopenia (HIT), a potentially fatal immune disorder affecting 1-5% of patients receiving heparin. Despite being a critically important element in HIT etiology, relatively little is known about the specific molecular mechanism of PF4-heparin interactions. This work uses native mass spectrometry to investigate PF4 interactions with relatively short heparin chains (up to decasaccharides). The protein is shown to be remarkably unstable at physiological ionic strength in the absence of polyanions; only monomeric species are observed, and the extent of multiple charging of corresponding ions indicates a partial loss of conformational integrity. The tetramer signal remains at or below the detection threshold in the mass spectra until the solution's ionic strength is elevated well above the physiological level, highlighting the destabilizing role played by electrostatic interactions vis-à-vis quaternary structure of this high-pI protein. The tetramer assembly is dramatically facilitated by relatively short polyanions (synthetic heparin-mimetic pentasaccharide), with the majority of the protein molecules existing in the tetrameric state even at physiological ionic strength. Each tetramer accommodates up to six pentasaccharides, with at least three such ligands required to guarantee the higher-order structure integrity. Similar results are obtained for PF4 association with longer and structurally heterogeneous heparin oligomers (decamers). These longer polyanions can also induce PF4 dimer assembly when bound to the protein in relatively low numbers, lending support to a model of PF4/heparin interaction in which the latter wraps around the protein, making contacts with multiple subunits. Taken together, these results provide a more nuanced picture of PF4-glycosaminoglycan interactions leading to complex formation. This work also advocates for a greater utilization of native mass spectrometry in elucidating molecular mechanisms underlying HIT, as well as other physiological processes driven by electrostatic interactions.

The Utility of Native MS for Understanding the Mechanism of Action of Repurposed Therapeutics in COVID-19: Heparin as a Disruptor of the SARS-CoV-2 Interaction with Its Host Cell Receptor.

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over 6,000,000 cases and nearly 400,000 deaths reported worldwide by the end of May 2020. A rush to find a cure prompted re-evaluation of a range of existing therapeutics vis-à-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the noncovalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease in its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19.

Identification of Protein Recognition Elements within Heparin Chains Using Enzymatic Foot-Printing in Solution and Online SEC/MS.

Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce the structural complexity by searching for "binding epitopes" within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. A top-down approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the longer and structurally diverse chains to interact with the client protein. Enzymatic lysis of the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with online mass spectrometry detection (SEC/MS) reveals the oligomers that are protected from lysis due to their tight association with the protein, and enables their characterization (both the oligomer length, and the number of incorporated sulfate and acetyl groups). When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels. The extent of sulfation of the minimal-length binder (hexamer) is relatively modest yet persistent, consistent with the notion of six sulfate groups being both essential and sufficient for antithrombin binding. However, the masses of longer surviving chains indicate complete sulfation of disaccharides beyond the hexasaccharide core. Molecular dynamics simulations confirm the existence of favorable electrostatic interactions between the high charge-density saccharide residues flanking the "canonical" antithrombin-binding hexasaccharide and the positive patch on the surface of the overall negatively charged protein. Furthermore, electrostatics may rescue the heparin/protein interaction in the absence of the canonical binding element.

Evaluation of top-down mass spectrometry and ion-mobility spectroscopy as a means of mapping protein-binding motifs within heparin chains.

Identifying structural elements within heparin (as well as other glycosaminoglycan) chains that enable their interaction with a specific client protein remains a challenging task due to the high degree of both intra- and inter-chain heterogeneity exhibited by this polysaccharide. The new experimental approach explored in this work is based on the assumption that the heparin chain segments bound to the protein surface will be less prone to collision-induced dissociation (CID) in the gas phase compared to the chain regions that are not involved in binding. Facile removal of the unbound chain segments from the protein/heparin complex should allow the length and the number of sulfate groups within the protein-binding segment of the heparin chain to be determined by measuring the mass of the truncated heparin chain that remains bound to the protein. Conformational integrity of the heparin-binding interface on the protein surface in the course of CID is ensured by monitoring the evolution of collisional cross-section (CCS) of the protein/heparin complexes as a function of collisional energy. A dramatic increase in CCS signals the occurrence of large-scale conformational changes within the protein and identifies the energy threshold, beyond which relevant information on the protein-binding segments of heparin chains is unlikely to be obtained. Testing this approach using a 1 : 1 complex formed by a recombinant form of an acidic fibroblast growth factor (FGF-1) and a synthetic pentasaccharide GlcNS,6S-GlcA-GlcNS,3S,6S-IdoA2S-GlcNS,6S-Me as a model system indicated that a tri-saccharide fragment is the minimal-length FGF-binding segment. Extension of this approach to a decameric heparin chain (dp10) allowed meaningful binding data to be obtained for a 1 : 1 protein/dp10 complex, while the ions representing the higher stoichiometry complex (2 : 1) underwent dissociation via asymmetric charge partitioning without generating truncated heparin chains that remain bound to the protein.

Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N terminus, alters helix dynamics, and reduces DNA affinity.

RcnR, a transcriptional regulator in Escherichia coli, derepresses the expression of the export proteins RcnAB upon binding Ni(II) or Co(II). Lack of structural information has precluded elucidation of the allosteric basis for the decreased DNA affinity in RcnR's metal-bound states. Here, using hydrogen-deuterium exchange coupled with MS (HDX-MS), we probed the RcnR structure in the presence of DNA, the cognate metal ions Ni(II) and Co(II), or the noncognate metal ion Zn(II). We found that cognate metal binding altered flexibility from the N terminus through helix 1 and modulated the RcnR-DNA interaction. Apo-RcnR and RcnR-DNA complexes and the Zn(II)-RcnR complex exhibited similar 2H uptake kinetics, with fast-exchanging segments located in the N terminus, in helix 1 (residues 14-24), and at the C terminus. The largest difference in 2H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N terminus and His-3, and has been associated with cognate metal binding. 2H uptake in helix 1 was suppressed in the Ni(II)- and Co(II)-bound RcnR complexes, in particular in the peptide corresponding to residues 14-24, containing Arg-14 and Lys-17. Substitution of these two residues drastically affected DNA-binding affinity, resulting in rcnA expression in the absence of metal. Our results suggest that cognate metal binding to RcnR orders its N terminus, decreases helix 1 flexibility, and induces conformational changes that restrict DNA interactions with the positively charged residues Arg-14 and Lys-17. These metal-induced alterations decrease RcnR-DNA binding affinity, leading to rcnAB expression.

Exploiting His-Tags for Absolute Quantitation of Exogenous Recombinant Proteins in Biological Matrices: Ruthenium as a Protein Tracer.

Metal labeling and ICP MS detection offer an alternative to commonly accepted techniques that are currently used to quantitate exogenous proteins in vivo, but modifying the protein surface with metal-containing groups inevitably changes its biophysical properties and is likely to affect trafficking and biodistribution. The approach explored in this work takes advantage of the presence of hexa-histidine tags in many recombinant proteins, which have high affinity toward a range of metals. While many divalent metals bind to poly histidine sequences reversibly, oxidation of imidazole-bound CoII or RuII is known to result in a dramatic increase of the binding strength. In order to evaluate the feasibility of using imidazole-bound metal oxidation as a means of attaching permanent tags to polyhistidine segments, a synthetic peptide YPDFEDYWMKHHHHHH was used as a model. RuII can be oxidized under ambient (aerobic) conditions, allowing any oxidation damage to the peptide beyond the metal-binding site to be avoided. The resulting peptide-RuIII complex is very stable, with the single hexa-histidine segment capable of accommodating up to three metal ions. Localization of RuIII within the hexa-histidine segment of the peptide was confirmed by tandem mass spectrometry. The RuIII/peptide binding appears to be irreversible, with both low- and high-molecular weight biologically relevant scavengers failing to strip the metal from the peptide. Application of this protocol to labeling a recombinant form of an 80 kDa protein transferrin allowed RuIII to be selectively placed within the His-tag segment. The metal label remained stable in the presence of ubiquitous scavengers and did not interfere with the receptor binding, while allowing the protein to be readily detected in serum at sub-nM concentrations. The results of this work suggest that ruthenium lends itself as an ideal metal tag for selective labeling of His-tag containing recombinant proteins to enable their sensitive detection and quantitation with ICP MS.

LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS.

Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection had been initially implemented using reversed phase LC, which in the past three decades remained the default type of chromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of other types of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to be preserved throughout the separation process, enjoyed relatively little appreciation until recently. However, the past few years witnessed a dramatic surge of interest in the so-called "native" (with "non-denaturing" being perhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysical communities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ion exchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are the benefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ion chemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers) and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography).

Mass Spectrometry Reveals a Multifaceted Role of Glycosaminoglycan Chains in Factor Xa Inactivation by Antithrombin.

Factor Xa (fXa) inhibition by antithrombin (AT) enabled by heparin or heparan sulfate is critical for controlling blood coagulation. AT activation by heparin has been investigated extensively, while interaction of heparin with trapped AT/fXa intermediates has received relatively little attention. We use native electrospray ionization mass spectrometry to study the role of heparin chains of varying length [hexa-, octa-, deca-, and eicosasaccharides (dp6, dp8, dp10, and dp20, respectively)] in AT/fXa complex assembly. Despite being critical promoters of AT/Xa binding, shorter heparin chains are excluded from the final products (trapped intermediates). However, replacement of short heparin segments with dp20 gives rise to a prominent ionic signal of ternary complexes. These species are also observed when the trapped intermediate is initially prepared in the presence of a short oligoheparin (dp6), followed by addition of a longer heparin chain (dp20), indicating that binding of heparin to AT/fXa complexes takes place after the inhibition event. The importance of the heparin chain length for its ability to associate with the trapped intermediate suggests that the binding likely occurs in a bidentate fashion (where two distinct segments of oligoheparin make contacts with the protein components, while the part of the chain separating these two segments is extended into solution to minimize electrostatic repulsion). This model is corroborated by both molecular dynamics simulations with an explicit solvent and ion mobility measurements in the gas phase. The observed post-inhibition binding of heparin to the trapped AT/fXa intermediates hints at the likely role played by heparan sulfate in their catabolism.

Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography.

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using ß2-microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule).

Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry.

Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.