Charged Residue Model Research Articles

Molecular Dynamics Simulations of Native Protein Charging via Proton Transfer during Electrospray Ionization with Grotthuss Diffuse H3O.

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.

Inter-Domain Repulsion of Dumbbell-Shaped Calmodulin during Electrospray Ionization Revealed by Molecular Dynamics Simulations.

The mechanisms whereby protein ions are released from nanodroplets at the liquid-gas interface have continued to be controversial since electrospray ionization (ESI) mass spectrometry was widely applied in biomolecular structure analysis in solution. Several viable pathways have been proposed and verified for single-domain proteins. However, the ESI mechanism of multi-domain proteins with more complicated and flexible structures remains unclear. Herein, dumbbell-shaped calmodulin was chosen as a multi-domain protein model to perform molecular dynamics simulations to investigate the structural evolution during the ESI process. For [Ca4CAM], the protein followed the classical charge residue model. As the inter-domain electrostatic repulsion increased, the droplet was found to split into two sub-droplets, while stronger-repulsive apo-calmodulin unfolded during the early evaporation stage. We designated this novel ESI mechanism as the domain repulsion model, which provides new mechanistic insights into further exploration of proteins containing more domains. Our results suggest that greater attention should be paid to the effect of domain-domain interactions on structure retention during liquid-gas interface transfer when mass spectrometry is used as the developing technique in gas phase structural biology.

Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility-Mass Spectrometry and Theory.

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge-dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: β-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term "native-like" to describe results from native ion mobility-mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.

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.

Using covalent modifications to distinguish protein electrospray mechanisms: Charged residue model (CRM) vs. chain ejection model (CEM)

Different mechanisms have been proposed for the formation of gaseous protein ions during electrospray ionization (ESI). In the charged residue model (CRM) ions are produced upon nanodroplet evaporation to dryness. This mechanism is thought to dominate in native ESI, where proteins retain compact conformations, with charge states close to the Rayleigh charge of protein-sized aqueous droplets. Much higher charge states are generated from proteins that are unfolded in solution. The chain ejection model (CEM) has been proposed for ESI under such denaturing conditions. In the CEM proteins are gradually expelled, while mobile H+ equilibrate between the droplet and its protruding tail. Providing clear-cut evidence for these scenarios remains difficult, because electrosprayed ions do not usually retain any features that reveal their formation mechanism. In this work we propose that the stepwise elimination of basic sites can serve to distinguish between the CRM and CEM. Using cytochrome c as a model system, we studied proteins that had between zero and 19 Lys blocked by acetylation. In native ESI (pH 7) the same low charge states were observed regardless of acetylation. This behavior is consistent with the CRM, where charge states are governed by protein size, rather than protein surface chemistry. Denaturing (pH 2) conditions resulted in much higher ESI charge states. Intriguingly, spectra acquired under these pH 2 conditions gradually shifted to lower charge states when the number of acetylated Lys was increased. This charge reduction is attributed to the fact that lowering the number of basic sites compromises the ability of the protein to compete with the droplet for mobile H+ during the CEM. In conclusion, we illustrate that simple covalent modifications can help distinguish between protein ion formation via the CRM or the CEM.

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.

Sample Flow Rate Scan in Electrospray Ionization Mass Spectrometry Reveals Alterations in Protein Charge State Distribution.

Sample flow rate is one of the parameters that influence the sensitivity of electrospray ionization (ESI) mass spectrometry. By varying the sample flow rate, initial droplets of different sizes can be generated. Protein molecules in small droplets may form gas-phase ions earlier than the ones in large droplets. Here, we have systematically studied the influence of sample flow rate on the ESI charge state distributions (CSDs) of model proteins. A dedicated programmable sample flow rate scanner was used to infuse protein samples at different flow rates into a mass spectrometer. The synergistic influence of sample flow rate and various electrolytes (ammonium acetate, ammonium bicarbonate, ammonium formate, and piperidine) was studied. Significant alterations to the CSDs with increasing flow rate were observed. For example, in the presence of ammonium acetate, at low flow rates, lower charge states of proteins showed high intensities, while at high flow rates, ions related to higher charge states of proteins dominated the spectra. On the other hand, in the presence of piperidine, a significant reduction in the ion currents of all charge states was observed during the flow rate scan. Our observations suggest that at low flow rates the protein molecules follow a charged residue model of ionization mechanism, and at high flow rates-due to structural changes in protein molecules in large ESI droplets-the charged residue and chain ejection models can possibly coexist. We propose the use of sample flow rate scan as a way to reveal the influence of flow rate on the CSDs of the studied proteins.

Current State of Oligonucleotide Characterization Using Liquid Chromatography-Mass Spectrometry: Insight into Critical Issues.

As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.

Formation of Gaseous Proteins via the Ion Evaporation Model (IEM) in Electrospray Mass Spectrometry.

The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na+. However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime.

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.

A Comparative Study for Sonic Spray and Electrospray Ionization Methods to Determine Noncovalent Protein–Ligand Interactions

A comparative study between sonic spray ionization (SSI) and electrospray ionization (ESI) is reported for cluster formation of proteins and ligands. For the observation of cluster formation, the interactions were not classified as specific or nonspecific and the instrumental parameters were held constant for both ion sources. Although ESI is a soft ionization method, it induces the formation of additional cluster formation between the protein–ligand couples. To understand this phenomenon, similar experiments were conducted using the same conditions by SSI. The equilibrium constants (Kd) of these complexes were calculated from the measurements obtained using both SSI and ESI. It is a common belief that SSI follows the charge residue model (CRM) and should form higher numbers of clusters. In the selected cases, although CRM is mostly followed, it does not give the same results in some specific situations. ESI produces more clusters during the ionization process for cytochrome-c maltoheptaose. The dependences of concentration, voltage and pH upon the complex formation were determined and number of maltoheptaose molecules bound were compared for both SSI and ESI sources. The main reason for the higher cluster formation is the applied voltage to the ESI source. High or low voltage causes distortion of globular structure of the proteins that leads to the formation of more clusters in the ESI source.

Chain Ejection Model for Electrospray Ionization of Unfolded Proteins: Evidence from Atomistic Simulations and Ion Mobility Spectrometry.

The ion evaporation model (IEM) and the charged residue model (CRM) represent cornerstones of any discussion related to the mechanism of electrospray ionization (ESI). Molecular dynamics (MD) simulations have confirmed that small ions such as Na+ are ejected from the surface of aqueous ESI droplets (IEM), while folded proteins in native ESI are released by water evaporation to dryness (CRM). ESI of unfolded proteins yields [M + zH] z+ ions that are much more highly charged than their folded counterparts. A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions (Konermann, L., et al. Anal. Chem. 2013, 85, 2-9). The CEM envisions that unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, followed by gradual ejection via intermediates where droplets carry extended protein tails. Thus far, it has not been possible to support the CEM through MD simulations using realistic protein models and atomistic force fields. Such endeavors require much larger droplets than in previous MD studies. Also, the incorporation of CEM-related H+ migration is difficult. This work overcomes these challenges in MD simulations on unfolded apo-myoglobin (aMb) in droplets with a 5.5 nm radius (∼22500 water molecules). We focused on solutions at pH ∼4 where the aMb solution charge coincides with the charge on some of the electrosprayed ions (22+ to 27+), such that H+ migration could be neglected. Na+ ions were added to ensure a droplet charge close to the Rayleigh limit. We found that 16 of 17 MD runs on various protonation patterns produced [M + zH] z+ ions via chain ejection. The predicted stretched-out aMb conformations were consistent with experimental collision cross sections. These results support the view that unfolded proteins follow the CEM. Overall, the IEM/CRM/CEM triad can account for a wide range of ESI scenarios involving various types of analytes.

Electrospray Ionization of Polypropylene Glycol: Rayleigh-Charged Droplets, Competing Pathways, and Charge State-Dependent Conformations.

Recent molecular dynamics (MD) simulations from various laboratories have advanced the general understanding of electrospray ionization (ESI)-related processes. Unfortunately, computational cost has limited most of those previous endeavors to ESI droplets with radii of ∼3 nm or less, which represent the low end of the size distribution in the ESI plume. The current work extends this range by conducting simulations on aqueous ESI droplets with radii of 5.5 nm (∼23 000 water molecules). Considering that computational cost increases with r6, this is a significant step forward. We focused on the ESI process for polypropylene glycol (PPG) which is a common ESI-MS calibrant. Different chain lengths (PPG10, 30, and 60) were tested in droplets that were charged with excess Na+. Solvent evaporation and Na+ ejection, with occasional progeny droplet formation, kept the systems at 80-100% of the Rayleigh limit throughout their life cycle. PPG chains migrated to the droplet surface where they captured Na+ via binding to ether oxygens. Various possible pathways for PPG release into the gas phase were encountered. Some PPG10 runs showed ejection from the droplet surface, consistent with the ion evaporation model (IEM). In other instances, PPG was released after near-complete solvent evaporation, as envisioned by the charged residue model (CRM). A third avenue was the partial separation from the droplet to form double or single-tailed structures, with subsequent chain detachment from the droplet. This last pathway is consistent with the chain ejection model (CEM). Immediately after detachment many chains were electrostatically stretched, but they subsequently collapsed into compact conformers. Extended structures were retained only for the most highly charged ions. Our simulations were complemented by ESI-MS and ion mobility measurements. MD-predicted charge states and collision cross sections were in agreement with these experimental data, supporting the mechanistic insights obtained.

How to run molecular dynamics simulations on electrospray droplets and gas phase proteins: Basic guidelines and selected applications.

The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.

Supercharging Proteins: How Many Charges Can a Protein Carry?

Very highly charged proteins, so-called "supercharged" ions, can lose (excess) protons to background gases like N2 . It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N2 but also much higher-basicity solvents. What mechanism(s) can explain such high charging, and what is the ultimate limit?

Mechanism of Protein Supercharging by Sulfolane and m-Nitrobenzyl Alcohol: Molecular Dynamics Simulations of the Electrospray Process.

Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets. Simulations were conducted on myoglobin in water, water/sulfolane, and water/m-NBA. Na(+) ions served as surrogate charge carriers instead of H(+). We focus on conditions where the protein initially adopts its native conformation. MD-generated charge states show remarkable agreement with experimental data. Droplet shrinkage is accompanied by Na(+) ejection, consistent with the ion evaporation model (IEM). The droplets segregate into an outer SCA shell and an aqueous core. This core harbors protein and Na(+). Unfavorable SCA solvation restricts Na(+) access to the droplet surface, thereby impeding IEM ejection. Rapid water loss causes SCA enrichment, ultimately forcing all remaining Na(+) to bind the protein. IEM ejection is no longer feasible after this point, such that the protein becomes supercharged by Na(+) trapping. SCA-free droplets produce lower charge states because the aqueous environment ensures a higher IEM efficiency. For all scenarios examined here, proteins are released via solvent evaporation to dryness, as envisioned by the charged residue model. Our data provide the first atomistic view of the supercharging mechanism.

Molecular Dynamics simulations of the electrospray process: formation of NaCl clusters via the charged residue mechanism.

Electrospray ionization (ESI) produces desolvated ions from solution phase analytes for mass spectrometric detection. The final steps of gas phase ion formation from nanometer-sized solvent droplets remain a matter of debate. According to the ion evaporation model (IEM), analytes are ejected from the droplet surface via field emission, whereas the charged residue model (CRM) envisions that ions are released upon droplet evaporation to dryness. Exposure of salt solutions to ESI conditions produces a range of cluster ions. Despite the rich literature on these systems, it is still unclear if these salt clusters form via the CRM or the IEM. The current study explores the formation of Na(n)Cl(m)((n-m)+) clusters from aqueous sodium chloride solution under positive and negative polarity conditions. Molecular dynamics (MD) methods are used for simulating the temporal evolution of charged NaCl-containing water droplets. A trajectory stitching approach is developed for continuously removing evaporated moieties from the simulation, thereby dramatically reducing computational cost. In addition, this procedure ensures adequate temperature control and eliminates evaporative cooling that would otherwise slow down the process. Continuous water evaporation leads to progressive droplet shrinkage, while the emission of solvated single ions ensures that the system remains at ca. 90% of the Rayleigh limit. Early during the process all ions in the droplet behave as freely dissolved species, but after a few nanoseconds at 370 K the systems gradually morph into amorphous wet salt aggregates. Ultimately, free Na(n)Cl(m)((n-m)+) clusters form as the last solvent molecules evaporate. Our data therefore provide direct evidence that sodium chloride cluster formation during ESI proceeds via the CRM. The IEM nonetheless plays an ancillary role, as it allows the system to shed charge (mostly in the form of hydrated Na(+) or Cl(-)) during droplet shrinkage. It appears that this study marks the first successful MD simulation of complete CRM processes.

Charge detection mass spectrometry of bacteriophage P22 procapsid distributions above 20 MDa.

Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids. P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution. The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges. The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa.

Effects of Polarity on the Structures and Charge States of Native-Like Proteins and Protein Complexes in the Gas Phase

Native mass spectrometry and ion mobility spectrometry were used to investigate the gas-phase structures of selected cations and anions of proteins and protein complexes with masses ranging from 6 to 468 kDa. Under the same solution conditions, the average charge states observed for all native-like anions were less than those for the corresponding cations. Using an rf-confining drift cell, similar collision cross sections were measured in positive and negative ion mode suggesting that anions and cations have very similar structures. This result suggests that for protein and protein complex ions within this mass range, there is no inherent benefit to selecting a specific polarity for capturing a more native-like structure. For peptides and low-mass proteins, polarity and charge-state dependent structural changes may be more significant. The charged-residue model is most often used to explain the ionization of large macromolecules based on the Rayleigh limit, which defines the upper limit of charge that a droplet can hold. Because ions of both polarities have similar structures and the Rayleigh limit does not depend on polarity, these results cannot be explained by the charged-residue model alone. Rather, the observed charge-state distributions are most consistent with charge-carrier emissions during the final stages of analyte desolvation, with lower charge-carrier emission energies for anions than the corresponding cations. These results suggest that the observed charge-state distributions in most native mass spectrometry experiments are determined by charge-carrier emission processes; although the Rayleigh limit may determine the gas-phase charge states of larger species, e.g., virus capsids.

Classification of the ejection mechanisms of charged macromolecules from liquid droplets

The relation between the charge state of a macromolecule and its ejection mechanism from droplets is one of the important questions in electrospray ionization methods. In this article, effects of solvent-solute interaction on the manifestation of the charge induced instability in a droplet are examined. We studied the instabilities in a prototype system of a droplet comprised of charged poly(ethylene glycol) and methanol, acetonitrile, and water solvents. We observed instances of three, previously only conjectured, [S. Consta, J. Phys. Chem. B 114, 5263 (2010)] mechanisms of macroion ejection. The mechanism of ejection of charged macroion in methanol is reminiscent of "pearl" model in polymer physics. In acetonitrile droplets, the instability manifests through formation of solvent spines around the solvated macroion. In water, we find that the macroion is ejected from the droplet through contiguous extrusion of a part of the chain. The difference in the morphology of the instabilities is attributed to the interplay between forces arising from the macroion solvation energy and the surface energy of the droplet interface. For the contiguous extrusion of a charged macromolecule from a droplet, we demonstrate that the proposed mechanism leads to ejection of the macromolecule from droplets with sizes well below the Rayleigh limit. The ejected macromolecule may hold charge significantly higher than that suggested by prevailing theories. The simulations reveal new mechanisms of macroion evaporation that differ from conventional charge residue model and ion evaporation mechanisms.