Mechanism of Protein Charging and Supercharging in Electrospray Ionization: Molecular Dynamics Simulations and Experimental Investigations

Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique for investigating protein structures, conformations, and interactions. Despite its widespread use, many fundamental aspects of ESI remain poorly understood. In this thesis, we use a combination of molecular dynamics (MD) simulations and experiments to gain insights into the hidden complexities of ESI-MS. Chapter 2 discusses the topic of salt-induced protein signal degradation. Salts such as NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl) interfere with MS data acquisition, leading to adduct formation and signal suppression. MD simulations provide an explanation for these salt interferences. Signal suppression can be broken down into two effects, i.e., i) peak splitting due to adduction, ii) “genuine” signal suppression. The results obtained may be helpful to anticipate solution conditions for improved protein analyses by ESI-MS. The two subsequent Chapters examine the mechanism of native protein supercharging, which represents a highly contentious topic. Chapter 3 uses MD simulations along with ion mobility mass spectrometry (IMS/MS). Holo-myoglobin (hMb) serves as a model protein, along with the two most common supercharging agents (SCAs), sulfolane and m-nitrobenzyl alcohol (m-NBA). Our data show that supercharging is caused by ‘charge trapping’ that arises from solvent segregation in the droplets, resulting in the formation of SCA-enriched surface layer and an aqueous core. The key factor to charge trapping is the differential solubility of charge carriers (such as Na+ or NH4+) in water compared to the exterior SCA layer. After complete water evaporation, residual SCA molecules impede charge carrier release from the droplet, and any remaining charge carriers will bind to the protein. Slow SCA evaporation eventually releases a highly charged protein into the gas phase that may undergo Coloumbic unfolding. These findings represent the first atomistic view of protein supercharging. In Chapter 4, we explore the mechanism of native protein supercharging from a different perspective using a crown ether (18C6). 18C6 selectively binds Na+/NH4+ and enhances their solubility in the SCA layer. This facilitates the release of 18C6-bound charge carriers from the droplet. As a result, 18C6 suppressed supercharging effect, as confirmed both in MD simulations and experimentally. These data support the proposed charge trapping mechanism for both proteins and dendrimers. A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions. 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. In Chapter 5 we overcome these difficulties and use MD simulations along with ion mobility experiments to confirm CEM as an ejection mechanism for unfolded proteins. Overall, the modeling and experimental work in this thesis provides unprecedented insights into the mechanism of protein charging and supercharging during ESI

Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging.

Native electrospray ionization (ESI) mass spectrometry (MS) aims to transfer proteins from solution into the gas phase while maintaining solution-like structures and interactions. The ability to control the charge states of protein ions produced in these experiments is of considerable importance. Supercharging agents (SCAs) such as sulfolane greatly elevate charge states without significantly affecting the protein structure in bulk aqueous solution. The origin of native ESI supercharging remains contentious. According to one model, SCAs trigger unfolding within ESI droplets. In contrast, the charge trapping model envisions that SCAs impede the ejection of charge carriers (e.g., NH4+ or Na+) from the droplet. We addressed this controversy experimentally and computationally by employing 18C6 crown ether as a mechanistic probe in native ESI-MS experiments on holo-myoglobin. Remarkably, 18C6 suppressed the supercharging capability of sulfolane. Molecular dynamics (MD) simulations reproduced the experimental charge states. The MD data revealed that 18C6 altered the location of charge carriers in the ESI droplets. Without 18C6, sulfolane covered the droplets in an ionophobic layer that impeded charge carrier access to the surface. In contrast, 18C6 complexation caused charge carrier enrichment in this surface layer, thereby promoting charge ejection. For late droplets, all the water had left and the protein was encapsulated in sulfolane; charge ejection at this stage continued only in the presence of 18C6. As a result, evaporation to dryness of charge-depleted water/sulfolane/18C6 droplets produced low protein charge states, whereas charge-abundant water/sulfolane droplets generated high charge states. Our data support the view that native ESI supercharging is caused by charge trapping. Unfolding within the droplet may play an ancillary role under some conditions, but for the cases examined here, protein structural changes are not a causative factor for supercharging. Our conclusions are bolstered by dendrimer supercharging experiments

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

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

Mechanism of Electrospray Supercharging for Unfolded Proteins: Solvent-Mediated Stabilization of Protonated Sites During Chain Ejection.

Proteins that are unfolded in solution produce higher charge states during electrospray ionization (ESI) than their natively folded counterparts. Protein charge states can be further increased by the addition of supercharging agents (SCAs) such as sulfolane. The mechanism whereby these supercharged [M + zH] z+ ions are formed under unfolded conditions remains unclear. Here we employed a combination of mass spectrometry (MS), ion mobility spectrometry (IMS), and molecular dynamics (MD) simulations for probing the ESI mechanism under denatured supercharging conditions. ESI of acid-unfolded apo-myoglobin (aMb) in the presence of sulfolane produced charge states around 27+, all the way to fully protonated (33+) aMb. MD simulations of aMb 27+ to 33+ in Rayleigh-charged water/sulfolane droplets culminated in electrostatically driven protein expulsion, consistent with the chain ejection model (CEM). The electrostatically stretched conformations predicted by these simulations were in agreement with IMS experiments. The CEM involves partitioning of mobile H+ between the droplet and the departing protein. Our results imply that supercharging of unfolded proteins is caused by residual sulfolane that stabilizes protonated sites on the protruding chains, thereby promoting H+ retention on the protein. The stabilization of charged sites is due to charge-dipole interactions mediated by the large dipole moment and the low volatility of sulfolane. Support for this mechanism comes from the experimental observation of sulfolane adducts on the most highly charged ions, a phenomenon previously noted by Venter ( J. Am. Soc. Mass Spectrom. 2012, 23, 489-497). The CEM supercharging model proposed here for unfolded proteins is distinct from the charge trapping mechanism believed to be operative during native ESI supercharging

Charging and supercharging of proteins for mass spectrometry: recent insights into the mechanisms of electrospray ionization.

Electrospray ionization (ESI) is an essential technique for transferring proteins from solution into the gas phase for mass spectrometry and ion mobility spectrometry. The mechanisms whereby [M + zH]z+ protein ions are released from charged nanodroplets during ESI have been controversial for many years. Here we discuss recent computational and experimental studies that have shed light on many of the mysteries in this area. Four types of protein ESI experiments can be distinguished, each of which appears to be associated with a specific mechanism. (i) Native ESI proceeds according to the charged residue model (CRM) that entails droplet evaporation to dryness, generating compact protein ions in low charge states. (ii) Native ESI supercharging is also a CRM process, but the dried-out proteins accumulate additional charge because supercharging agents such as sulfolane interfere with the ejection of small ions (Na+, NH4+, etc.) from the shrinking droplets. (iii) Denaturing ESI follows the chain ejection model (CEM), where protein ions are gradually expelled from the droplet surface. H+ equilibration between the droplets and the protruding chains culminates in highly charged gaseous proteins, analogous to the collision-induced dissociation of multi-protein complexes. (iv) Denatured ESI supercharging also generates protein ions via the CEM. Supercharging agents stabilize protonated sites on the protein tail via charge-dipole interactions, causing the chain to acquire additional charge. There will likely be scenarios that fall outside of these four models, but it appears that the framework outlined here covers most of the experimentally relevant conditions

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

Charging and supercharging of proteins for mass spectrometry: Recent insights into the mechanisms of electrospray ionization

peer reviewe

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 <i>r</i>⁶, 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

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 + <i>z</i>H]^<i>z</i>+ 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. <i>Anal. Chem</i>. <b>2013</b>, <i>85</i>, 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 + <i>z</i>H]^<i>z</i>+ 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