RESUMEN
Carbohydrates have various biological functions that are based on their structures. However, the composition and the glycosidic-bond linkage and configuration of carbohydrates present challenges for their characterization. Furthermore, isomeric features contribute to the formation of intramolecular hydrogen bonds, which influence the flexibility and dynamics of carbohydrates. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) enables the analysis of protein dynamics by monitoring deuterium labeling after HDX for different lengths of time. In-electrospray ionization (in-ESI) HDX-MS has been used to rapidly label solvated carbohydrates with labeling occurring during desolvation of ESI droplets. Therefore, HDX-labeling times can be altered by changing the spray-solvent conductivity, which changes the initial size of ESI droplets and their resulting lifetimes. Here, we utilize in-ESI HDX-MS to characterize nine isomeric disaccharides with different monosaccharide compositions and glycosidic-bond linkages and configurations. We compared both the relative D-uptake of isomers at individual conductivities, or HDX-labeling times, and the trends associated with labeling at multiple conductivities. Interestingly, the relative D-uptake trends were correlated to isomeric features that affect disaccharide flexibility, including formation of intramolecular hydrogen bonds. Among the isomeric features studied, linkage was observed to have a significant influence on relative D-uptake with (1-3)-linked disaccharides having more change in relative D-uptake with changing conductivity compared to other linkages. Overall, this research illustrates how in-ESI HDX-MS can be applied to structurally characterize disaccharides with distinct isomeric features. Furthermore, this work shows that in-ESI HDX-MS can be used to monitor the dynamics of solvated molecules with rapidly exchanging functional groups.
Asunto(s)
Disacáridos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Disacáridos/química , Solventes/química , Enlace de Hidrógeno , IsomerismoRESUMEN
The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples.
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Unraveling the mechanism by which native proteins are charged through electrospray ionization (ESI) has been the focus of considerable research because observable charge states can be correlated to biophysical characteristics, such as protein folding and, thus, solution conformation. Difficulties in characterizing electrosprayed droplets have catalyzed the use of molecular dynamics (MD) to provide insights into the mechanisms by which proteins are charged and transferred to the gas phase. However, prior MD studies have utilized metal ions, primarily Na+, as charge carriers, even though proteins are primarily detected as protonated ions in the mass spectra. Here, we propose a modified MD protocol for simulating discrete Grotthuss diffuse H3O+ that is capable of dynamically altering amino-acid protonation states to model electrospray charging and gaseous ion formation of model proteins, ubiquitin, and myoglobin. Application of the protocol to the evaporation of acidic droplets enables a molecular perspective of H3O+ coordination and proton transfer to/from proteins, which is unfeasible with the metal charge carriers used in previous MD studies of ESI. Our protocol recreates experimentally observed charge-state distributions and supports the charge residue model (CRM) as the dominant mechanism of native protein ionization during ESI. Additionally, our results suggest that protonation is highly specific to individual residues and is correlated to the formation of localized hydrated regions on the protein surface as droplets desolvate. Considering the use of discrete H3O+ instead of Na+, the developed protocol is a necessary step toward developing a more comprehensive model of protein ionization during ESI.
Asunto(s)
Simulación de Dinámica Molecular , Protones , Espectrometría de Masa por Ionización de Electrospray/métodos , Mioglobina/química , Iones/química , Gases/químicaRESUMEN
Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray hydrogen/deuterium exchange mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid- and base-catalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes.
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Medición de Intercambio de Deuterio , Hidrógeno , Deuterio , Medición de Intercambio de Deuterio/métodos , Péptidos/química , Carbohidratos , Hexosas , Concentración de Iones de HidrógenoRESUMEN
Tandem mass spectrometry (MS/MS) using fragmentation has become one of the most effective methods for gaining sequence and structural information on biomolecules. Ion/ion reactions are competitive reactions, where either proton transfer (PT) or electron transfer (ET) can occur from interactions between multiply charged cations and singly charged anions. Utilizing ion/ion reactions with fluoranthene has offered a unique method of fragment formation for the structural elucidation of biomolecules. Fluoranthene is considered an ideal anion reagent because it selectively causes electron-transfer dissociation (ETD) and minimizes PT when interacting with peptides. However, limited investigations have sought to understand how fluorantheneâthe primary, commercially available anion reagentâinteracts with other biomolecules. Here, we apply deuterium labeling to investigate ion/ion reaction mechanisms between fluoranthene and divalent, metal-adducted carbohydrates (Ca2+, Mg2+, Co2+, and Ni2+). Deuterium labeling of carbohydrates allowed us to observe evidence of hydrogen/deuterium exchange (HDX) occurring after ion/ion dissociation reactions. The extent of deuterium loss is dependent on several factors, including the physical properties of the metal ion and the fragment structure. Based on the deuterium labeling data, we have proposed ETD, PTD, and intermolecular PTâalso described as HDXâmechanisms. This research provides a fundamental perspective of ion/ion and ion/molecule reaction mechanisms and illustrates properties that impact ion/ion and ion/molecule reactions for carbohydrates. Together, this could improve the capability to distinguish complex and heterogeneous biomolecules, such as carbohydrates.
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Protones , Espectrometría de Masas en Tándem , Deuterio , Carbohidratos , Aniones , Medición de Intercambio de Deuterio/métodosRESUMEN
The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.
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Escherichia coli , Humanos , Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol O-Acetiltransferasa/metabolismo , Ligandos , Acetilcoenzima A , Espectrometría de Masas/métodos , Escherichia coli/química , TermodinámicaRESUMEN
Carbohydrates and glycans are integral to many biological processes, including cell-cell recognition and energy storage. However, carbohydrates are often difficult to analyze due to the high degree of isomerism present. One method being developed to distinguish these isomeric species is hydrogen/deuterium exchange-mass spectrometry (HDX-MS). In HDX-MS, carbohydrates are exposed to a deuterated reagent and the functional groups with labile hydrogen atoms, including hydroxyls and amides, exchange with the 1 amu heavier isotope, deuterium. These labels can then be detected by MS, which monitors the mass increase with the addition of D-labels. The observed rate of exchange is dependent on the exchanging functional group, the accessibility of the exchanging functional group, and the presence of hydrogen bonds. Herein, we discuss how HDX has been applied in the solution-phase, gas-phase, and during MS ionization to label carbohydrates and glycans. Additionally, we compare differences in the conformations that are labeled, the labeling timeframes, and applications of each of these methods. Finally, we comment on future opportunities for development and use of HDX-MS to analyze glycans and glycoconjugates.
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Carbohidratos , Hidrógeno , Hidrógeno/química , Deuterio/química , Amidas , HexosasRESUMEN
Glycans, carbohydrates, and glycoconjugates are involved in many crucial biological processes, such as disease development, immune responses, and cell-cell recognition. Glycans and carbohydrates are known for the large number of isomeric features associated with their structures, making analysis challenging compared with other biomolecules. Mass spectrometry has become the primary method of structural characterization for carbohydrates, glycans, and glycoconjugates. Metal adduction is especially important for the mass spectrometric analysis of carbohydrates and glycans. Metal-ion adduction to carbohydrates and glycoconjugates affects ion formation and the three-dimensional, gas-phase structures. Herein, we discuss how metal-ion adduction impacts ionization, ion mobility, ion activation and dissociation, and hydrogen/deuterium exchange for carbohydrates and glycoconjugates. We also compare the use of different metals for these various techniques and highlight the value in using metals as charge carriers for these analyses. Finally, we provide recommendations for selecting a metal for analysis of carbohydrate adducts and describe areas for continued research.
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Previous reports present different models for the stabilization of the Fc-FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (171MGKHRY176) of FcγRIa. Complexes formed from the FcγRIa receptor and IgG1s containing biantennary glycans with N-acetylglucosamine, galactose, and α2,6-N-acetylneuraminic terminations were measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS), classified for dissimilarity with Welch's ANOVA and Games-Howell post hoc procedures, and modeled with molecular dynamics (MD) simulations. For each glycoform of the IgG1-FcγRIa complex peptic peptides of Fab, Fc and FcγRIa report distinct H/D exchange rates. MD simulations corroborate the differences in the peptide deuterium content through calculation of the percent of time that transient glycan-peptide bonds exist. These results indicate that stability of IgG1-FcγRIa complexes correlate with the presence of intermolecular glycoprotein interactions between the IgG1 glycans and the 173KHR175 motif within the FG-loop of FcγRIa. The results also indicate that intramolecular glycan-protein bonds stabilize the Fc region in isolated and complexed IgG1. Moreover, HDX-MS data evince that the Fab domain has glycan-protein binding contacts within the IgG1-FcγRI complex.
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Complejo Antígeno-Anticuerpo/química , Glicoproteínas/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Inmunoglobulina G/química , Simulación de Dinámica Molecular , Receptores de IgG/química , Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/metabolismo , Galactosa , Glicoproteínas/metabolismo , Proteínas de la Membrana/química , Péptidos/química , Péptidos/metabolismo , Polisacáridos , Unión ProteicaRESUMEN
Electrospray ionization (ESI) is frequently used to produce gas-phase ions for mass spectrometry (MS)-based techniques. The composition of solvents used in ESI-MS is often manipulated to enhance analyte ionization, including for carbohydrates. Moreover, to characterize analyte structures, ESI has been coupled to hydrogen/deuterium exchange, ion mobility, and tandem MS. Therefore, it is important to understand how solvent composition affects the structure of carbohydrates during and after ESI. In this work, we use molecular dynamics to simulate the desolvation of ESI droplets containing a model carbohydrate and observe the formation of carbohydrate adducts with metal ions. Molecular-level details on the effects of formulating mixtures of water, methanol, and acetonitrile to achieve enhanced ionization are presented. We complement our simulations with ESI-MS experiments. We report that when sprayed from aqueous mixtures containing volatile solvents, carbohydrates ionize to form metal-ion adducts rapidly due to rapid solvent evaporation rather than changes in the ionization mechanism. We find that when sprayed from solvent mixtures, carbohydrates are primarily solvated by water due to the migration of more volatile solvents to the surface of the droplet. Ultimately, the structure of the carbohydrate varies depending on its solvent environment, as inter- and intramolecular interactions are affected. We propose that solvents with 25% or more water may be used to enhance the ionization of carbohydrates with minimal effect on the structure during and after ESI.
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Native mass spectrometry (MS) is used to elucidate the stoichiometry of protein complexes and quantify binding interactions by maintaining native-like, noncovalent interactions in the gas phase. However, ionization forces proteins into specific conformations, losing the solution-phase dynamics associated with solvated protein structures. Comparison of gas-phase structures to those in solution, or to other gas-phase ion populations, has many biological implications. For one, analyzing the variety of conformations that are maintained in the gas-phase can provide insight into a protein's solution-phase energy landscape. The gas-phase conformations of proteins and complexes can be investigated using ion mobility (IM) spectrometry. Specifically, drift tube (DT)-IM utilizes uniform electric fields to propel a population of gas-phase ions through a region containing a neutral gas. By measuring the mobility (K) of gas-phase ions, users are able to calculate an average momentum transfer cross section (DTCCS), which provides structural information on the ion. Conversely, in traveling-wave ion mobility spectrometry (TWIMS), TWCCS values cannot be derived directly from an ion's mobility but must be determined following calibration. Though the required calibration adds uncertainty, it is common to report only an average and standard deviation of the calculated TWCCS, accounting for uncertainty associated with replicate measurements, which is a fraction of the overall uncertainty. Herein, we calibrate a TWIMS instrument and derive TWCCSN2 and TWCCSN2âHe values for four proteins: cytochrome c, ubiquitin, apo-myoglobin, and holo-myoglobin. We show that compared to reporting only the standard deviation of TWCCS, propagating error through the calibration results in a significant increase in the number of calculated TWCCS values that agree within experimental error with literature values (DTCCS). Incorporating this additional uncertainty provides a more thorough assessment of a protein ion's gas-phase conformations, enabling the structures sampled by native IM-MS to be compared against other reported structures, both experimental and computational.
RESUMEN
Carbohydrates play key roles in facilitating cellular functions, yet characterizing their structures is analytically challenging due to the presence of epimers, regioisomers, and stereoisomers. In-electrospray-hydrogen/deuterium exchange-mass spectrometry (in-ESI HDX-MS) is a rapid HDX method that samples solvated carbohydrates with minimal instrument modification. When applied to proteins, HDX is often measured after multiple time points to sample the dynamics of structures. Herein, we alter the HDX reaction time by modifying the spray-solvent conductivity, which changes the initial size of ESI droplets, and thus, the droplet lifetimes. We show that this change in droplet lifetime alters the magnitude of HDX for carbohydrate-metal adducts. Furthermore, we illustrate how monitoring HDX at multiple time points enables three trisaccharide isomers (melezitose, maltotriose, and isomaltotriose) to be distinguished. This work illustrates the feasibility of this method for characterizing solvated carbohydrates, including isomeric species which differ only by linkage.
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Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is a well-established technique for structural analysis of proteins. In HDX experiments it is common to label for multiple, different lengths of time to characterize protein structures and dynamics. However, applications of HDX to carbohydrates have been limited due to the rapid exchange rates of hydroxyls, which have also prevented the development and application of methods that sample HDX at multiple timepoints. Theta capillaries pulled to electrospray tips have been used to achieve microsecond reaction times. Here, we report the utilization of theta-ESI emitters to achieve multiple timepoints for deuteration of carbohydrates. We increased the labeling time for HDX by increasing the initial ESI droplet sizes using theta-ESI emitters with increasing tip opening sizes. The reaction times achieved by varying the tip sizes ranged from sub-microsecond to â¼20 µs, with the average number of deuterium exchanges varying from 0.5 ± 0.2 D to 5 ± 3 D for sodium-adducted melezitose, which contains 11 labile hydrogens. Our findings are significant because this is the first report of carbohydrates analyzed by solution-phase HDX to achieve multiple H/D exchange timepoints.
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Trisacáridos/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Glycans have an immense number of biological activities, necessitating increased efforts to characterize glycan structures. Mass spectrometry has been coupled to electrospray ionization (ESI) to characterize carbohydrates. While the gas-phase structures of glycan- and carbohydrate-metal adducts have been characterized, several questions persist concerning the mechanism of transfer of carbohydrates from ESI droplets into the gas phase. Using various computational methods, including molecular dynamics, steered molecular dynamics, and density functional theory calculations, we present a mechanistic investigation on the evaporation of solvent from nanosized droplets, formation of carbohydrate-metal adducts, and their subsequent release into the gas phase. We relate the computational results to mass spectra of melezitose, a model carbohydrate, and its permethylated derivative. Our results confirm two mechanisms for the release of carbohydrate-ion adducts from solvated droplets. Native (unmodified) carbohydrates are ionized via the charged residue model, while the permethylated derivative is ionized via the ion evaporation model. For both mechanisms, the monomer carbohydrate-metal adduct is the dominant species observed. This work illustrates that the ionization mechanisms are dictated by interactions between the carbohydrate and solvent, and coordination of the carbohydrate with the metal ion. Thus, these results provide insight into the molecular interactions that govern the mechanism of release.
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Nanoestructuras/química , Sodio/química , Trisacáridos/química , Agua/química , Conformación de Carbohidratos , Teoría Funcional de la Densidad , Gases/química , Modelos Químicos , Simulación de Dinámica Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported â¼89â¯800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (â¼78â¯900 centroids), giving â¼100% coverage, and â¼10â¯900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σxÌ ). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.
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Anticuerpos Monoclonales/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Fragmentos Fab de Inmunoglobulinas/análisisRESUMEN
In-electrospray (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to characterize solvated carbohydrate structures. However, the rapid exchange rate of hydroxyls, as well as variations in source conditions and ambient humidity, alter the extent of forward and back exchange, resulting in poor repeatability when quantifying D-uptake on different days. Herein, we compare two internal standards, a peptide and derivatized carbohydrate, to improve the repeatability of in-ESI HDX of carbohydrate-metal adducts. Our results show that maltoheptaose, derivatized with Girard's T reagent, is a suitable internal standard for improving the repeatability of in-ESI HDX analyses of carbohydrates of varying size.
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Glycosylation is a post-translational modification that is often altered in disease development and progression, including cancer. In cancerous patients, the abnormal expression of glycosylation enzymes leads to aberrant glycosylation, which has been linked to malignant tissues. Due to aberrant glycosylation, the presence of specific glycans can be used as biomarkers for identifying the type and stage of cancer. Glycan structures are heterogeneous, with different protein glycoforms having different functional activities. Lectins are an important tool in glycan analysis due to their specificity in binding to unique glycan linkages and monosaccharide units, which allows for the identification of unique glycan structural properties. In this review, we will discuss the use of lectins in microarrays and chromatography for characterizing glycan structures.
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Bioensayo/métodos , Lectinas/metabolismo , Neoplasias/metabolismo , Polisacáridos/metabolismo , Biomarcadores , Cromatografía de Afinidad , Glicosilación , Humanos , Lectinas/química , Análisis por Micromatrices , Polisacáridos/químicaRESUMEN
The conformations of glycans are crucial for their biological functions. In-electrospray ionization (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a promising technique for studying carbohydrate conformations since rapidly exchanging functional groups, e.g., hydroxyls, can be labeled on the timeframe of ESI. However, regular application of in-ESI HDX to characterize carbohydrates requires further analysis of the in-ESI HDX methodology. For instance, in this method, HDX occurs concurrently to the analyte transitioning from solution to gas-phase ions. Therefore, there is a possibility of sampling both gas-phase and solution-phase conformations of the analyte. Herein, we differentiate in-ESI HDX of metal-adducted carbohydrates from gas-phase HDX and illustrate that this method analyzes solvated species. We also systematically examine the effects of ESI parameters, including spray solvent composition, auxiliary gas flow rate, sheath gas flow rate, sample infusion rate, sample concentration, and spray voltage, and discuss their effects on in-ESI HDX. Further, we model the structural changes of a trisaccharide, melezitose, and its intramolecular and intermolecular hydrogen bonding in solvents with different compositions of methanol and water. These molecular dynamic simulations support our experimental results and illustrate how an individual ESI parameter can alter the conformations we sample by in-ESI HDX. In total, this work illustrates how the fundamental processes of ESI alter the magnitude of HDX for carbohydrates and suggest parameters that should be considered and/or optimized prior to performing experiments with this in-ESI HDX technique. Graphical Abstract á .