Identifying and Quantifying Relative Concentrations of Epimers in Mixtures via Cyclic Ion Mobility Mass Spectrometry: Dexamethasone and Betamethasone as a Case Study.

Epimers can show different biological activities and different pharmacological behaviors; therefore, their separation and analysis are crucial in the drug development process. Due to their similar chemical and physical properties, separation of epimers is challenging. This study demonstrates the application of cyclic ion mobility-mass spectrometry to separate, identify, and quantify dexamethasone and betamethasone in a binary mixture. Cyclic IMS separation of the isolated protonated dimer resulted in three peaks: dexamethasone homodimer, betamethasone homodimer, and their heterodimer. Besides providing improved separation over the protonated monomer, the presence of a heterodimer peak provides additional confirmation of an isomeric mixture. We identified the dexamethasone and betamethasone homodimer peaks by infusing pure solutions of each epimer and measuring each pure homodimer's arrival time. The measured peak areas indicated that the heterodimer is formed at twice the rate of each homodimer and that dexamethasone and betamethasone contribute equally to the heterodimer signal. Using this observation, we could accurately calculate the relative concentrations of each epimer by adding half of the heterodimer peak area to each homodimer peak area. These findings enable the identification and quantification of dexamethasone and betamethasone based on the arrival time distributions of their protonated dimers. This is the first demonstration of accurate relative quantification of epimers by separating charged dimers in the gas phase.

Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

An alphacoronavirus polymerase structure reveals conserved replication factor functions.

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine structures of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. One structure shows an unexpected nsp8 stoichiometry despite remaining bound to RNA. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha- and betacoronaviruses. Our work demonstrates the importance of studying diverse coronaviruses in revealing aspects of coronavirus replication and identifying areas of conservation to be targeted by antiviral drugs.

Mass spectrometers as cryoEM grid preparation instruments.

Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air-water interface often leads to sample damage and preferential orientation of the particles. Using native mass spectrometry to create cryoEM samples, potentially avoids these problems and allows the use of mass spectrometry sample isolation techniques during EM grid creation. We review the recent publications that have demonstrated protein complexes can be ionized, flown through the mass spectrometer, gently landed onto EM grids, imaged, and reconstructed in 3D. Although many uncertainties and challenges remain, the combination of cryoEM and MS has great potential.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

An alphacoronavirus polymerase structure reveals conserved co-factor functions.

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.

Onto Grid Purification and 3D Reconstruction of Protein Complexes Using Matrix-Landing Native Mass Spectrometry.

Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.

Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes.

Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexes─GroEL, the 20S proteasome core particle, and ß-galactosidase─and obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (∼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.

Three-dimensional structure determination of protein complexes using matrix-landing mass spectrometry.

Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe the use of a chemical landing matrix that preserves the structural integrity of the deposited particles. With this system we obtain a three-dimensional reconstruction of the 800 kDa protein complex GroEL from gas-phase deposited GroEL ions. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

Infrared Photoactivation Boosts Reporter Ion Yield in Isobaric Tagging.

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.

Characterization of Homopolymer Distributions via Direct Infusion ESI-MS/MS using Wide Mass-to-Charge Windows and Gas-Phase Ion/Ion Reactions.

A hallmark of electrospray ionization (ESI) of large polymeric molecules is its tendency to generate charge state distributions. When a distribution of polymers is subjected to ESI, the charge state distribution of each component can lead to a mass spectrum composed of a highly congested mixture of ions with overlapping mass-to-charge (m/z) ratios. When the polymers are composed of a common monomeric unit (i.e., a homopolymer), the overlap of the charge state distributions of the polymer components can give rise to striking spectral patterns with a dense central cluster of peaks having similar m/z values and wing-like patterns on either side. We refer to the central cluster of peaks as an "Emerald City," with a nod to the Wizard of Oz, combining the wings as an "Emerald City pattern". The Emerald City pattern can appear in the mass spectrum of any homopolymer with distributions of charge states and sizes. Various parameters were studied individually for their contributions to the appearance of Emerald City patterns. Dextran samples were used to demonstrate the spectral pattern experimentally, and a web-based tool was developed to validate the findings. We also proposed to use direct infusion ESI-MS coupled with segmented m/z windows that encompass Emerald Cities followed by gas-phase proton transfer reactions for characterizing poly disperse synthetic polymer samples. Poly(ethylenimine) samples were used as model systems to demonstrate the approach. The proposed strategy improves sample characterization relative to conventional zero-charge deconvolution or proton transfer reactions without prior mass-selected m/z windows.

Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry.

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either ß-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.

Multivariable analysis of the influence of cross-reactive carbohydrate determinant inhibition and other factors on intradermal and serological allergen test results: a prospective, multicentre study.

BACKGROUND: Serological allergen testing (SAT) is used widely to formulate allergen-specific immunotherapy for atopic dogs. Serum immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) can produce false-positive reactions, creating discrepancy between SAT and intradermal allergen test (IDAT) results. OBJECTIVES: The primary objective was to determine if inhibition of anti-CCD IgE in a commercial assay improved correlation with IDAT. The secondary objective was to assess the influence of dog- and clinic-specific factors, environmental factors, putative allergen exposure and prior medications on intradermal and SAT reactivity. ANIMALS: Two-hundred and eleven client-owned dogs were enrolled from eight North American dermatology specialty practices. METHODS AND MATERIALS: Collection of serum samples and IDAT were performed on the same day. Sera were assayed for detection of IgE specific to 25 allergens, before and after treatment with a proprietary inhibitor of anti-CCD IgE. Data for each dog were collected via a questionnaire filled out by veterinary personnel. RESULTS: The correlation between the testing modalities was fair before (Spearman's rho, ρ = 0.2092) and after (ρ = 0.3042) inhibition of anti-CCD IgE. Ciclosporin dose (P = 0.003), independent of duration of use, and duration of lokivetmab use (P = 0.001), independent of dose administered, were associated with statistically significant decreases in IgE concentrations across all allergen types. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to previous reports, this study demonstrated unchanged correlation between SAT and IDAT after inhibition of anti-CCD IgE. Ciclosporin dose and lokivetmab treatment duration may have unexplored effects on IgE concentration during SAT.

Detection and inhibition of IgE antibodies reactive with cross-reactive carbohydrate determinants in an ELISA for allergen-specific IgE in horses.

BACKGROUND: It has been demonstrated that immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) is present in the serum of sensitized humans, dogs and cats, and that these CCD-specific antibodies might confound serological testing. HYPOTHESIS/OBJECTIVE: The objective was to determine whether or not CCD-reactive antibodies occur in horses and to investigate the prevalence of CCD-reactive IgE antibodies in equine sera using a monoclonal cocktail-based enzyme-linked immunosorbent assay designed to detect allergen-specific IgE in horses, and to evaluate a means for successful inhibition of these CCD. METHODS AND MATERIALS: Sera from 28 horses suspected of clinical allergy were evaluated, with and without a proprietary inhibitor which contains carbohydrates derived from bromelain (BROM-CCD), using a panel of 72 allergens that include 15 grasses, 17 trees, nine weeds, five mites, 12 fungi, 12 insects and two environmental allergens. RESULTS: Twenty-five samples were shown to be reactive to at least one of the allergens, and 15 were reactive to 10 allergens or more. BROM-CCD had minimal effect on the mite reactivity in any of the positive samples; however, substantial inhibition for pollen allergens (trees, grasses and weeds) was demonstrable. Reduction in signal to pollens ranged from 20% to 100% for samples that were inhibited by CCD-BROM. CONCLUSIONS AND CLINICAL IMPORTANCE: These results demonstrate that CCD-reactive IgE antibodies are evident in horses and that BROM-CCD can be effective in reducing reactions with these irrelevant carbohydrates and will likely yield a more accurate in vitro allergen reactivity profile for selection of allergens included in an immunotherapeutic regime.

Mass Analysis of Macro-molecular Analytes via Multiply-Charged Ion Attachment.

A novel gas-phase charge and mass manipulation approach is demonstrated to facilitate the mass measurement of high mass complexes within the context of native mass spectrometry. Electrospray ionization applied to solutions generated under native or near-native conditions has been demonstrated to be capable of preserving biologically relevant complexes into the gas phase as multiply charged ions suitable for mass spectrometric analysis. However, charge state distributions tend to be narrow and extensive salt adduction, heterogeneity, and so on tend to lead to significantly broadened peaks. These issues can compromise mass measurement of high mass bio-complexes, particularly when charge states are not clearly resolved. In this work, we show that the attachment of high mass ions of known mass and charge to populations of ions of interest can lead to well-separated signals that can yield confident charge state and mass assignments from otherwise poorly resolved signals.

Digital ion trap mass analysis of high mass protein complexes using IR activation coupled with ion/ion reactions.

Native mass spectrometry (MS) focuses on measuring the masses of large biomolecular complexes and probing their structures. Large biomolecular complexes are readily introduced into mass spectrometers as gas-phase ions using electrospray ionization (ESI); however, the ions tend to be heavily adducted with solvent and salts, which leads to mass measurement errors. Various solution clean-up approaches can reduce the degree of adduction prior to introduction to the mass spectrometer. Gas-phase activation of trapped ions can provide additional adduct reduction, and charge reduction ion/ion reactions increase charge state separation. Together, gas-phase activation and charge reduction can combine to yield spectra of well separated charge states for improved mass measurements. A simple gas-phase collisional activation technique is to apply a dipolar DC (DDC) field to opposing electrodes in an ion trap. DDC activation loses its efficacy when ions are trapped at low q values, which is true of the high m/z ions generated by charge reduction ion/ion reactions. Digital ion trapping (DIT) readily traps high m/z ions at higher q values by varying trapping frequency rather than amplitude, but the low frequencies used to trap high m/z ions also decreases the efficacy of DDC activation. We demonstrate here using ions derived from GroEL that IR activation of ions shows no discrimination against high m/z ions trapped with DIT, because they can be focused equally well to the trap center to interact with the IR laser beam. Following pump out of excess background gas, IR activation can also induce efficient dissociation of the GroEL complex. This work demonstrates that IR activation is an effective approach for ion heating in native MS over the unusually wide range of charge states accessible via gas-phase ion/ion reactions.

Detection and Inhibition of IgE for cross-reactive carbohydrate determinants evident in an enzyme-linked immunosorbent assay for detection of allergen-specific IgE in the sera of dogs and cats.

BACKGROUND: It has been demonstrated recently that immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) is present in the serum of allergen-sensitized dogs and cats, and that these CCD-specific antibodies might confound serological testing. HYPOTHESIS/OBJECTIVE: The objective was to document the prevalence of CCD detectable in a monoclonal cocktail-based enzyme-linked immunosorbent assay designed for the detection of allergen-specific IgE in the sera of dogs and cats, and to define a means for successful inhibition of these CCD. METHODS AND MATERIALS: The incidence of reactivity to bromelain and a commercially available inhibitor of carbohydrate-specific antibodies (RIDA-CCD) was evaluated in 100 dog sera samples before and after inhibition with RIDA-CCD and a proprietary inhibitor containing carbohydrates derived from bromelain (BROM-CCD). Subsequently, sera from 600 dogs and 600 cats were evaluated using a serum diluent with and without BROM-CCD. RESULTS: Both the RIDA-CCD and BROM-CCD inhibitors demonstrated successful reduction of CCD reactivity, although a more efficient profile of inhibition was evident with BROM-CCD. Mite reactivity in dog and cat sera was largely unaffected; however, substantial inhibition for pollen allergens (trees, grasses and weeds) was shown. After BROM-CCD inhibition, 1% of canine samples and 13% of feline samples were rendered completely negative for allergen reactivity. CONCLUSIONS AND CLINICAL IMPORTANCE: The results demonstrate that BROM-CCD is effective in reducing reactions with irrelevant carbohydrates, and that inhibition of CCD reactivity might substantially alter the outcome of the in vitro reactivity profile used for selection of allergens to be included in an immunotherapeutic regime.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions.

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high m/z ions. The additional tandem-MS (MSn) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at m/z > 50,000.