MD Simulations of Peptide-Containing Electrospray Droplets: Effects of Parameter Settings on the Predicted Mechanisms of Gas Phase Ion Formation.

Electrospray ionization (ESI) mass spectrometry is widely used for interrogating peptides, proteins, and other biomolecular analytes. A growing number of laboratories use molecular dynamics (MD) simulations for uncovering ESI mechanisms by modeling the behavior of highly charged nanodroplets. The outcome of any MD simulation depends on certain assumptions and parameter settings, and it is desirable to optimize these factors by benchmarking computational data against experiments. Unfortunately, benchmarking of ESI simulations is difficult because experimentally generated gaseous ions do not generally retain any features that would reveal their formation pathway [e.g., the charged residue mechanism (CRM) or the ion evaporation mechanism (IEM)]. Here, we tackle this problem by examining the effects of various MD settings on the ESI behavior of the 9-residue peptide bradykinin in acidic aqueous droplets. Several parameters were found to significantly affect the kinetic competition between peptide IEM and CRM. By systematically probing the droplet behavior, we uncovered problems associated with certain settings, including peptide/solvent temperature imbalances, unexpected peptide deceleration during IEM, and a dependence of the ESI mechanism on the water model. We also noted different simulation outcomes for different force fields. On the basis of comprehensive tests, we propose a set of "best practice" parameter settings for MD simulations of ESI droplets. The strategies used here should be transferable to other types of droplet simulations, paving the way toward a more solid understanding of ESI mechanisms.

Mechanism of Protein Aggregation Inhibition by Arginine: Blockage of Anionic Side Chains Favors Unproductive Encounter Complexes.

Aggregation refers to the assembly of proteins into nonphysiological higher order structures. While amyloid has been studied extensively, much less is known about amorphous aggregation, a process that interferes with protein expression and storage. Free arginine (Arg+) is a widely used aggregation inhibitor, but its mechanism remains elusive. Focusing on myoglobin (Mb), we recently applied atomistic molecular dynamics (MD) simulations for gaining detailed insights into amorphous aggregation (Ng J. Phys. Chem. B 2021, 125, 13099). Building on that approach, the current work for the first time demonstrates that MD simulations can directly elucidate aggregation inhibition mechanisms. Comparative simulations with and without Arg+ reproduced the experimental finding that Arg+ significantly decreased the Mb aggregation propensity. Our data reveal that, without Arg+, protein-protein encounter complexes readily form salt bridges and hydrophobic contacts, culminating in firmly linked dimeric aggregation nuclei. Arg+ promotes the dissociation of encounter complexes. These "unproductive" encounter complexes are favored because Arg+ binding to D- and E- lowers the tendency of these anionic residues to form interprotein salt bridges. Side chain blockage is mediated largely by the guanidinium group of Arg+, which binds carboxylates through H-bond-reinforced ionic contacts. Our MD data revealed Arg+ self-association into a dynamic quasi-infinite network, but we found no evidence that this self-association is important for protein aggregation inhibition. Instead, aggregation inhibition by Arg+ is similar to that mediated by free guanidinium ions. The computational strategy used here should be suitable for the rational design of aggregation inhibitors with enhanced potency.

On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry.

Ammonium acetate (NH4Ac) is a widely used solvent additive in native electrospray ionization (ESI) mass spectrometry. NH4Ac can undergo proton transfer to form ammonia and acetic acid (NH4+ + Ac- → NH3 + HAc). The volatility of these products ensures that electrosprayed ions are free of undesired adducts. NH4Ac dissolution in water yields pH 7, providing "physiological" conditions. However, NH4Ac is not a buffer at pH 7 because NH4+ and Ac- are not a conjugate acid/base pair (Konermann, L. J. Am. Soc. Mass Spectrom. 2017, 28, 1827-1835.). In native ESI, it is desirable that analytes experience physiological conditions not only in bulk solution but also while they reside in ESI droplets. Little is known about the internal milieu of NH4Ac-containing ESI droplets. The current work explored the acid/base chemistry of such droplets, starting from a pH 7 analyte solution. We used a two-pronged approach involving evaporation experiments on bulk solutions under ESI-mimicking conditions, as well as molecular dynamics simulations using a newly developed algorithm that allows for proton transfer. Our results reveal that during droplet formation at the tip of the Taylor cone, electrolytically generated protons get neutralized by Ac-, making NH4+ the net charge carriers in the weakly acidic nascent droplets. During the subsequent evaporation, the droplets lose water as well as NH3 and HAc that were generated by proton transfer. NH3 departs more quickly because of its greater volatility, causing the accumulation of HAc. Together with residual Ac-, these HAc molecules form an acetate buffer that stabilizes the average droplet pH at 5.4 ± 0.1, as governed by the Henderson-Hasselbalch equation. The remarkable success of native ESI investigations in the literature implies that this pH drop by ∼1.6 units relative to the initially neutral analyte solution can be tolerated by most biomolecular analytes on the short time scale of the ESI process.

Effects of Hydrogen/Deuterium Exchange on Protein Stability in Solution and in the Gas Phase.

Mass spectrometry (MS)-based techniques are widely used for probing protein structure and dynamics in solution. H/D exchange (HDX)-MS is one of the most common approaches in this context. HDX is often considered to be a "benign" labeling method, in that it does not perturb protein behavior in solution. However, several studies have reported that D2O pushes unfolding equilibria toward the native state. The origin, and even the existence of this protein stabilization remain controversial. Here we conducted thermal unfolding assays in solution to confirm that deuterated proteins in D2O are more stable, with 2-4 K higher melting temperatures than unlabeled proteins in H2O. Previous studies tentatively attributed this phenomenon to strengthened H-bonds after deuteration, an effect that may arise from the lower zero-point vibrational energy of the deuterated species. Specifically, it was proposed that strengthened water-water bonds (W···W) in D2O lower the solubility of nonpolar side chains. The current work takes a broader view by noting that protein stability in solution also depends on water-protein (W···P) and protein-protein (P···P) H-bonds. To help unravel these contributions, we performed collision-induced unfolding (CIU) experiments on gaseous proteins generated by native electrospray ionization. CIU profiles of deuterated and unlabeled proteins were indistinguishable, implying that P···P contacts are insensitive to deuteration. Thus, protein stabilization in D2O is attributable to solvent effects, rather than alterations of intraprotein H-bonds. Strengthening of W···W contacts represents one possible explanation, but the stabilizing effect of D2O can also originate from weakened W···P bonds. Future work will be required to elucidate which of these two scenarios is correct, or if both contribute to protein stabilization in D2O. In any case, the often-repeated adage that "D-bonds are more stable than H-bonds" does not apply to intramolecular contacts in native proteins.

Using Density Functional Theory for Testing the Robustness of Mobile-Proton Molecular Dynamics Simulations on Electrosprayed Ions: Structural Implications for Gaseous Proteins.

Current experiments only provide low-resolution information on gaseous protein ions generated by electrospray ionization (ESI). Molecular dynamics (MD) simulations can yield complementary insights. Unfortunately, conventional MD does not capture the mobile nature of protons in gaseous proteins. Mobile-proton MD (MPMD) overcomes this limitation. Earlier MPMD data at 300 K indicated that protein ions generated by "native" ESI retain solution-like structures with a hydrophobic core and zwitterionic exterior [Bakhtiari, M.; Konermann, L. J. Phys. Chem. B 2019, 123, 1784-1796]. MPMD redistributes protons using electrostatic and proton affinity calculations. The robustness of this approach has never been scrutinized. Here, we close this gap by benchmarking MPMD against density functional theory (DFT) at the B3LYP/6-31G* level, which is well suited for predicting proton affinities. The computational cost of DFT necessitated the use of small peptides. The MPMD energetic ranking of proton configurations was found to be consistent with DFT single-point energies, implying that MPMD can reliably identify favorable protonation sites. Peptide MPMD runs converged to DFT-optimized structures only when applying 300-500 K temperature cycling, which was necessary to prevent trapping in local minima. Temperature cycling MPMD was then applied to gaseous protein ions. Native ubiquitin converted to slightly expanded structures with a zwitterionic core and a nonpolar exterior. Our data suggest that such inside-out protein structures are intrinsically preferred in the gas phase, and that they form in ESI experiments after moderate collisional excitation. This is in contrast to native ESI (with minimal collisional excitation, simulated by MPMD at 300 K), where kinetic trapping promotes the survival of solution-like structures. In summary, this work validates the MPMD approach for simulations on gaseous peptides and proteins.

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations.

Peptide separations by reversed-phase liquid chromatography (RPLC) are an integral part of bottom-up proteomics. These separations typically employ C18 columns with water/acetonitrile gradient elution in the presence of formic acid. Despite the widespread use of such workflows, the exact nature of peptide interactions with the stationary and mobile phases is poorly understood. Here, we employ microsecond molecular dynamics (MD) simulations to uncover details of peptide RPLC. We examined two tryptic peptides, a hydrophobic and a hydrophilic species, in a slit pore lined with C18 chains that were grafted onto SiO2 support. Our simulations explored peptide trapping, followed by desorption and elution. Trapping in an aqueous mobile phase was initiated by C18 contacts with Lys butyl moieties. This was followed by extensive anchoring of nonpolar side chains (Leu/Ile/Val) in the C18 layer. Exposure to water/acetonitrile triggered peptide desorption in a stepwise fashion; charged sites close to the termini were the first to lift off, followed by the other residues. During water/acetonitrile elution, both peptides preferentially resided close to the pore center. The hydrophilic peptide exhibited no contacts with the stationary phase under these conditions. In contrast, the hydrophobic species underwent multiple transient Leu/Ile/Val binding interactions with C18 chains. These nonpolar interactions represent the foundation of differential peptide retention, in agreement with the experimental elution behavior of the two peptides. Extensive peptide/formate ion pairing was observed in water/acetonitrile, particularly at N-terminal sites. Overall, this work uncovers an unprecedented level of RPLC molecular details, paving the way for MD simulations as a future tool for improving retention prediction algorithms and for the design of novel column materials.

Mechanism of Magic Number NaCl Cluster Formation from Electrosprayed Water Nanodroplets.

Events taking place during electrospray ionization (ESI) can trigger the self-assembly of various nanoclusters. These products are often dominated by magic number clusters (MNCs) that have highly symmetrical structures. The literature rationalizes the dominance of MNCs by noting their high stability. However, this argument is not necessarily adequate because thermodynamics cannot predict the outcome of kinetically controlled reactions. Thus, the mechanisms responsible for MNC dominance remain poorly understood. Molecular dynamics (MD) simulations can provide atomistic insights into self-assembly reactions, but even this approach has thus far failed to provide pertinent answers. The current work overcomes this limitation. We focused on salt clusters formed from aqueous NaCl solutions during ESI. The corresponding mass spectra are dominated by the Na14Cl13+ MNC. Simulations of ESI droplets showed nonspecific association of Na+ and Cl-, culminating in gaseous clusters via solvent evaporation to dryness (charged residue mechanism). These nascent clusters did not show any preference for MNCs. In mass spectrometry experiments, analyte ions undergo in-source activation prior to detection. We emulated in-source activation by heating nascent clusters in our MD runs. Heating triggered structural fluctuations and dissociation events, generating MNC-dominated product distributions. Why are MNCs preferred after in-source activation? Thermally excited clusters frequently adopt structures consisting of a preformed MNC and a stringlike protrusion that contains the surplus ions. Facile separation of these protrusions releases the MNC (Clusterhot → MNC-protrusion → MNC + protrusion). This work marks the first time that MD simulations were able to capture cluster self-assembly with subsequent "molecular pruning", generating MNC-dominated product distributions that agree with experiments.

Structural Dynamics of a Thermally Stressed Monoclonal Antibody Characterized by Temperature-Dependent H/D Exchange Mass Spectrometry.

Differential scanning calorimetry (DSC) is a standard tool for probing the resilience of monoclonal antibodies (mAbs) and other protein therapeutics against thermal degradation. Unfortunately, DSC usually only provides insights into global unfolding, although sequential steps are sometimes discernible for multidomain proteins. Temperature-dependent hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential to probe heat-induced events at a much greater level of detail. We recently proposed a strategy to deconvolute temperature-dependent HDX data into contributions from local dynamics, global unfolding/refolding, as well as chemical labeling. However, that strategy was validated only for a small protein (Tajoddin, N. N.; Konermann, L. Anal. Chem. 2020, 92, 10058). The current work explores the applicability of this HDX framework to the NIST reference mAb (NISTmAb), a large multidomain protein that is prone to aggregation and has three melting points. Using global fitting, we were able to model HDX profiles across the NISTmAb sequence between zero and 95 °C, and for time points between 15 s and 20 min. We uncovered the enthalpic and entropic contributions of local fluctuations that govern the conformational dynamics at low temperatures. The CH2 and CH3 domains were found to be increasingly affected by global unfolding/refolding in the vicinity of their melting points, although the transiently unfolded protein displayed significant residual protection. Global dynamics were not involved in the deuteration of the Fab domains (which have the highest melting point). Instead, global Fab unfolding was followed immediately by irreversible aggregation. Our results reveal that the thermodynamic HDX-MS strategy applied in this work is well suited for probing spatially resolved dynamics of thermally stressed large proteins such as mAbs, complementing data obtained by DSC.

Grotthuss Molecular Dynamics Simulations for Modeling Proton Hopping in Electrosprayed Water Droplets.

Excess protons in water exhibit unique transport properties because they can rapidly hop along H-bonded water wires. Considerable progress has been made in unraveling this Grotthuss diffusion mechanism using quantum mechanical-based computational techniques. Unfortunately, high computational cost tends to restrict those techniques to small systems and short times. Molecular dynamics (MD) simulations can be applied to much larger systems and longer time windows. However, standard MD methods do not permit the dissociation/formation of covalent bonds, such that Grotthuss diffusion cannot be captured. Here, we bridge this gap by combining atomistic MD simulations (using Gromacs and TIP4P/2005 water) with proton hopping. Excess protons are modeled as hydronium ions that undergo H3O+ + H2O → H2O + H3O+ transitions. In accordance with ab initio MD data, these Grotthuss hopping events are executed in "bursts" with quasi-instantaneous hopping across one or more waters. The bursts are separated by regular MD periods during which H3O+ ions undergo Brownian diffusion. The resulting proton diffusion coefficient agrees with the literature value. We apply this Grotthuss MD technique to highly charged water droplets that are in a size regime encountered during electrospray ionization (5 nm radius, ∼17,000 H2O). The droplets undergo rapid solvent evaporation and occasional H3O+ ejection, keeping them at ca. 81% of the Rayleigh limit. The simulated behavior is consistent with phase Doppler anemometry data. The Grotthuss MD technique developed here should be useful for modeling the behavior of various proton-containing systems that are too large for high-level computational approaches. In particular, we envision future applications related to electrospray processes, where earlier simulations used metal cations while in reality excess protons dominate.

Formation of Gaseous Peptide Ions from Electrospray Droplets: Competition between the Ion Evaporation Mechanism and Charged Residue Mechanism.

The transfer of peptide ions from solution into the gas phase by electrospray ionization (ESI) is an integral component of mass spectrometry (MS)-based proteomics. The mechanisms whereby gaseous peptide ions are released from charged ESI nanodroplets remain unclear. This is in contrast to intact protein ESI, which has been the focus of detailed investigations using molecular dynamics (MD) simulations and other methods. Under acidic liquid chromatography/MS conditions, many peptides carry a solution charge of 3+ or 2+. Because of this pre-existing charge and their relatively small size, prevailing views suggest that peptides follow the ion evaporation mechanism (IEM). The IEM entails analyte ejection from ESI droplets, driven by electrostatic repulsion between the analyte and droplet. Surprisingly, recent peptide MD investigations reported a different behavior, that is, the release of peptide ions via droplet evaporation to dryness which represents the hallmark of the charged residue mechanism (CRM). Here, we resolved this conundrum by performing MD simulations on a common model peptide (bradykinin) in Rayleigh-charged aqueous droplets. The primary focus was on pH 2 conditions (bradykinin solution charge = 3+), but we also verified that our MD strategy captured pH-dependent charge state shifts seen in ESI-MS experiments. In agreement with earlier simulations, we found that droplets with initial radii of 1.5-3 nm predominantly release peptide ions via the CRM. In contrast, somewhat larger radii (4-5 nm) favor IEM behavior. It appears that these are the first MD data to unequivocally demonstrate the viability of peptide IEM events. Electrostatic arguments can account for the observed droplet size dependence. In summary, both CRM and IEM can be operative in peptide ESI-MS. The prevalence of one over the other mechanism depends on the droplet size distribution in the ESI plume.

Mechanism of Thermal Protein Aggregation: Experiments and Molecular Dynamics Simulations on the High-Temperature Behavior of Myoglobin.

Proteins that encounter unfavorable solvent conditions are prone to aggregation, a phenomenon that remains poorly understood. This work focuses on myoglobin (Mb) as a model protein. Upon heating, Mb produces amorphous aggregates. Thermal unfolding experiments at low concentration (where aggregation is negligible), along with centrifugation assays, imply that Mb aggregation proceeds via globally unfolded conformers. This contrasts studies on other proteins that emphasized the role of partially folded structures as aggregate precursors. Molecular dynamics (MD) simulations were performed to gain insights into the mechanism by which heat-unfolded Mb molecules associate with one another. A prerequisite for these simulations was the development of a method for generating monomeric starting structures. Periodic boundary condition artifacts necessitated the implementation of a partially immobilized water layer lining the walls of the simulation box. Aggregation simulations were performed at 370 K to track the assembly of monomeric Mb into pentameric species. Binding events were preceded by multiple unsuccessful encounters. Even after association, protein-protein contacts remained in flux. Binding was mediated by hydrophobic contacts, along with salt bridges that involved hydrophobically embedded Lys residues. Overall, this work illustrates that atomistic MD simulations are well suited for garnering insights into protein aggregation mechanisms.

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c.

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrome c (cyt c). Compared to the Fe(II) protein, Fe(III) cyt c shows enhanced deuteration on both the distal and proximal sides of the heme. Previous studies have attributed the enhanced dynamics of Fe(III) cyt c to the facile and reversible rupture of the distal M80-Fe(III) bond. Using molecular dynamics (MD) simulations, we conducted a detailed analysis of various cyt c conformers. Our MD data confirm that rupture of the M80-Fe(III) contact triggers major reorientation of the distal Ω loop. Surprisingly, this event takes place with only miniscule H-bonding alterations. In other words, the distal loop dynamics are almost "HDX-silent". Moreover, distal loop movements cannot account for enhanced dynamics on the opposite (proximal) side of the heme. Instead, enhanced deuteration of Fe(III) cyt c is attributed to sparsely populated conformers where both the distal (M80) and proximal (H18) coordination bonds have been ruptured, along with opening of numerous H-bonds on both sides of the heme. We conclude that there can be major structural fluctuations that are only weakly coupled to changes in H-bonding, making them virtually impossible to track by HDX-MS. In such cases, HDX-MS may provide an incomplete view of protein dynamics.

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization.

Native electrospray ionization (ESI)-mass spectrometry (MS) is widely used for the detection and characterization of multi-protein complexes. A well-known problem with this approach is the possible occurrence of nonspecific protein clustering in the ESI plume. This effect can distort the results of binding affinity measurements, and it can even generate gas-phase complexes from proteins that are strictly monomeric in bulk solution. By combining experiments and molecular dynamics (MD) simulations, the current work for the first time provides detailed insights into the ESI clustering of proteins. Using ubiquitin as a model system, we demonstrate how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific clusters (e.g., dimers or trimers) via solvent evaporation to dryness. These events are in line with earlier proposals, according to which protein clustering is associated with the charged residue model (CRM). MD simulations on cytochrome c (which carries a large intrinsic positive charge) confirmed the viability of this CRM avenue. In addition, the cytochrome c data uncovered an alternative mechanism where protein-protein contacts were formed early within ESI droplets, followed by cluster ejection from the droplet surface. This second pathway is consistent with the ion evaporation model (IEM). The observation of these IEM events for large protein clusters is unexpected because the IEM has been thought to be associated primarily with low-molecular-weight analytes. In all cases, our MD simulations produced protein clusters that were stabilized by intermolecular salt bridges. The MD-generated charge states agreed with experiments. Overall, this work reveals that ESI-induced protein clustering does not follow a tightly orchestrated pathway but can proceed along different avenues.

Nrf2, the Major Regulator of the Cellular Oxidative Stress Response, is Partially Disordered.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.

Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of Electrosprayed Protein Ions.

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH+/A- contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H+ are mobile, such that H+ transfer can convert salt bridges into neutral B0/HA0 contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH+/A- versus B0/HA0 interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H+ have dissociation energies around 700 kJ mol-1. The corresponding B0/HA0 contacts are 1 order of magnitude weaker. When considering the effects of mobile H+, BH+/A- bond energies were found to be between these two extremes, confirming that H+ migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H+ did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H+ resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H+ simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H+ limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H+ in MD simulations will result in unrealistic outcomes under CIU conditions.

Sulfolane-Induced Supercharging of Electrosprayed Salt Clusters: An Experimental/Computational Perspective.

It is well-known that supercharging agents (SCAs) such as sulfolane enhance the electrospray ionization (ESI) charge states of proteins, although the mechanistic origins of this effect remain contentious. Only very few studies have explored SCA effects on analytes other than proteins or peptides. This work examines how sulfolane affects electrosprayed NaI salt clusters. Such alkali metal halide clusters have played a key role for earlier ESI mechanistic studies, making them interesting targets for supercharging investigations. ESI of aqueous NaI solutions predominantly generated singly charged [NanI(n-1)]+ clusters. The addition of sulfolane resulted in abundant doubly charged [NanI(n-2)Sulfolanes]2+ species. These experimental data for the first time demonstrate that electrosprayed salt clusters can undergo supercharging. Molecular dynamics (MD) simulations of aqueous ESI nanodroplets containing Na+/I- with and without sulfolane were conducted to obtain atomistic insights into the supercharging mechanism. The simulations produced [NanIi]z+ and [NanIiSulfolanes]z+ clusters similar to those observed experimentally. The MD trajectories demonstrated that these clusters were released into the gas phase upon droplet evaporation to dryness, in line with the charged residue model. Sulfolane was found to evaporate much more slowly than water. This slow evaporation, in conjunction with the large dipole moment of sulfolane, resulted in electrostatic stabilization of the shrinking ESI droplets and the final clusters. Hence, charge-dipole stabilization causes the sulfolane-containing droplets and clusters to retain more charge, thereby providing the mechanistic foundation of salt cluster supercharging.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Interrogating the Quaternary Structure of Noncanonical Hemoglobin Complexes by Electrospray Mass Spectrometry and Collision-Induced Dissociation.

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, "noncanonical" hexameric and octameric complexes were observed. CID of all these species [(αß)2, (αß)3, and (αß)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes.

Enhancing Protein Electrospray Charge States by Multivalent Metal Ions: Mechanistic Insights from MD Simulations and Mass Spectrometry Experiments.

The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La3+ boosts protein charge states relative to monovalent metals (e.g., Na+). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na+, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La3+ showed a very different behavior. The trivalent nature of La3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.