Native SEC and Reversed-Phase LC-MS Reveal Impact of Fab Glycosylation of Anti-SARS-COV-2 Antibodies on Binding to the Receptor Binding Domain.

The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (∼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.

Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry.

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the Stabilitome.

The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an Escherichia coli lysate is provided to extend this approach to characterize the thermal stability of a proteome. As the solution temperature is increased, proteins and protein complexes undergo structural and organizational transitions; for each species, the folded ↔ unfolded and assembled ↔ disassembled populations are monitored based on changes in vT-ESI-MS charge state distributions and masses. The robustness of the approach illustrates a step toward the proteome-wide characterization of thermal stabilities and structural transitions-the stabilitome.

Understanding the Thermal Denaturation of Myoglobin with IMS-MS: Evidence for Multiple Stable Structures and Trapped Pre-equilibrium States.

Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.0) was monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the stability and investigate structural changes involved in unfolding. We utilize two experimental approaches to induce thermal denaturation: a variable-temperature electrospray ionization (vT-ESI) source that heats the bulk solution in the ESI emitter, and a variable-power 10.6 µm CO2 laser that rapidly heats nanodroplets produced by ESI. These two approaches sample different time scales of the denaturation process; long time scales (seconds to minutes) where the system is at equilibrium using the vT-ESI approach and shorter time scales (µs) by rapid droplet heating in which the system is in a pre-equilibrium state. Increasing the solution temperature (from 28 to 95 °C in the vT-ESI experiments) shifts the charge state distribution from low charge states ([M + 7H]7+ to [M + 9H]9+) to more highly charged species. This is accompanied by loss of the heme group to yield the apomyoglobin (aMb) species, indicating that the protein has unfolded. Monitoring the formation of aMb and the shift in average charge states of aMb and hMb with solution temperature allows for relative quantitation of their individual stabilities, highlighting the stabilizing effects of heme binding. We compare the degree of unfolding induced by heating the bulk solution (using vT-ESI) to the laser droplet heating approach and find that the rapid nature of the laser heating approach allows for transient pre-equilibrium states to be sampled.

Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions.

Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS "snapshot" approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Melting of Hemoglobin in Native Solutions as measured by IMS-MS.

Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to ∼40 °C), a heterotetrameric (α2ß2) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo αß forms) before ultimately forming insoluble aggregates at higher temperatures (>60 °C). In addition to the holo αß forms, a small population of αß dimers containing only a single heme ligand and having a dioxidation modification mapping to the ß subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys93 residue of the ß subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the αß subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).

Characterizing Thermal Transitions of IgG with Mass Spectrometry.

Variable temperature electrospray ionization (ESI) is coupled with mass spectrometry techniques in order to investigate structural transitions of monoclonal antibody immunoglobulin G (IgG) in a 100-mM ammonium acetate (pH 7.0) solution from 26 to 70 °C. At 26 °C, the mass spectrum for intact IgG shows six charge states + 22 to + 26. Upon increasing the solution temperature, the fraction of low-charge states decreases and new, higher-charge state ions are observed. Upon analysis, it appears that heating the solution aids in desolvation of the intact IgG precursor. Above ~ 50 °C, a cleavage event between the light and heavy chains is observed. An analysis of the kinetics for these processes at different temperatures yields transition state thermochemistry of ΔH‡ = 95 ± 10 kJ mol-1, ΔS‡ = 8 ± 1 J mol-1 K-1, and ΔG‡ = 92 ± 11 kJ mol-1. The mechanism for light chain dissociation appears to involve disulfide bond scrambling that ultimately results in a non-native Cys199-Cys217 disulfide bond in the light chain product. Above ~ 70 °C, we are unable to produce a stable ESI signal. The loss of signal is ascribed to aggregation that is primarily associated with the remaining portion of the antibody after having lost the light chain. Graphical Abstract.

Variable-Temperature ESI-IMS-MS Analysis of Myohemerythrin Reveals Ligand Losses, Unfolding, and a Non-Native Disulfide Bond.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.

Substance P in the Gas Phase: Conformational Changes and Dissociations Induced by Collisional Activation in a Drift Tube.

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H]3+ ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way, it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H]3+ in the absence of solvent are on the order of ~ 40 kJ mol-1, substantially lower than the ~ 90 kJ mol-1 required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited. Graphical Abstract.

Conformationally Regulated Peptide Bond Cleavage in Bradykinin.

Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H]2++H+ → [BK+3H]3+, that is regulated by trans → cis isomerization of Arg1-Pro2, resulting in the Arg1- cis-Pro2- cis-Pro3-Gly4-Phe5-Ser6- cis-Pro7-Phe8-Arg9 (all- cis) configuration. Once formed, the all- cis [BK+3H]3+ spontaneously cleaves the bond between Pro2-Pro3 with perfect specificity, a bond that is biologically resistant to cleavage by any human enzyme. Temperature-dependent kinetics studies reveal details about the intrinsic peptide processing mechanism. We propose that nonenzymatic cleavage at Pro2-Pro3 occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg1-Pro2. From this mechanism, we can extract transition state thermochemistry: Δ G‡ = 94.8 ± 0.2 kJ·mol-1, Δ H‡ = 79.8 ± 0.2 kJ·mol-1, and Δ S‡ = -50.4 ± 1.7 J·mol-1·K-1 for the trans → cis protonation event; and, Δ G‡ = 94.1 ± 9.2 kJ·mol-1, Δ H‡ = 107.3 ± 9.2 kJ·mol-1, and Δ S‡ = 44.4 ± 5.1 J·mol-1·K-1 for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro3-Gly4-Phe5-Ser6- cis-Pro7-Phe8-Arg9 that is approximately an order of magnitude more antigenic than BK.

Melting proteins confined in nanodroplets with 10.6 µm light provides clues about early steps of denaturation.

Ubiquitin confined within nanodroplets was irradiated with a variable-power CO2 laser. Mass spectrometry analysis shows evidence for a protein "melting"-like transition within droplets prior to solvent evaporation and ion formation. Ion mobility spectrometry reveals that structures associated with early steps of denaturation are trapped because of short droplet lifetimes.

Melting Proteins: Evidence for Multiple Stable Structures upon Thermal Denaturation of Native Ubiquitin from Ion Mobility Spectrometry-Mass Spectrometry Measurements.

Ion mobility and mass spectrometry techniques are coupled with a temperature-controlled electrospray ionization source to follow the structural transitions of ubiquitin in aqueous solution (pH = 3) at elevated solution temperatures (T = 26-96 °C). Changes in the charge state distribution are consistent with a two-state, cooperative unfolding transition having a melting temperature of Tm = 71 ± 2 °C, in agreement with prior measurements [ Wintrode , P. L. ; Makhatadze , G. I. ; Privalov , P. L. Proteins , 1994 , 18 , 246 - 253 ]. However, analysis of ion mobility distributions reveals the two-state transition is a composite of transitions involving at least nine unique species: three native or native-like structures; two that appear to be equilibrium intermediates (i.e., populations of new conformers that form at elevated temperatures but subsequently disappear at higher temperatures); and four products observed at high temperatures, including the well-characterized ubiquitin A state, and two solution species that are differentiated based on a cis- or trans-configured Glu18-Pro19 peptide bond. These nine states vary in abundances by factors as large as ∼103 over the range of solution temperatures. Although experimental melting transitions are conceived as a loss of well-defined structure leading to a random distribution of unstructured, denatured forms, the results provide evidence for new conformers having at least some well-defined structural elements are stabilized as temperature is increased.

Rapid high mass resolution mass spectrometry using matrix-assisted ionization.

Matrix-assisted ionization (MAI) is demonstrated to be a robust and sensitive analytical method capable of analyzing proteins such as cholera toxin B-subunit and pertussis toxin mutant from conditions containing relatively high amounts of inorganic salts, buffers, and preservatives without the need for prior sample clean-up or concentration. By circumventing some of the sample preparation steps, MAI simplifies and accelerates the analytical workflow for biological samples in complex media. The benefits of multiply charged ions characteristic of electrospray ionization (ESI) and the robustness of matrix-assisted laser desorption/ionization (MALDI) can be obtained from a single method, making it well suited for analysis of proteins and other biomolecules at ultra-high resolution as demonstrated on an Orbitrap Fusion where protein subunits were resolved for which MALDI-time-of-flight failed. MAI results are compared with those obtained with ESI, MALDI, and laserspray ionization methods and fundamental commonalities discussed.

Reproducibility and quantification of illicit drugs using matrix-assisted ionization (MAI) mass spectrometry.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

High-throughput characterization of small and large molecules using only a matrix and the vacuum of a mass spectrometer.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.