Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

RATIONALE: Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. METHODS: The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. RESULTS: Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. CONCLUSION: Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

SpectraX: A Straightforward Tool for Principal Component Analysis-Based Spectral Analysis.

The analysis of complex spectra is an important component of direct/ambient mass spectrometry (MS) applications such as natural product screening. Unlike chromatography-based metabolomics or proteomics approaches, which rely on software and algorithms, the work of spectral screening is mostly performed manually in the initial stages of research and relies heavily on the experience of the analyst. As a result, throughput and spectral screening reliability are problematic when dealing with large amounts of data. Here, we present SpectraX, a MATLAB-based application, which can analyze MS spectra and quickly locate m/z features from them. Principal component analysis (PCA) is used to analyze the data set, and scoring plots are presented to help in understanding the clustering of data. The algorithm uses mass to charge (m/z) features to produce a list of potential natural products.

3D printer platform and conductance feedback loop for automated imaging of uneven surfaces by liquid microjunction-surface sampling probe mass spectrometry.

RATIONALE: Molecular imaging of samples using mass spectrometric techniques, such as matrix-assisted laser desorption ionization or desorption electrospray ionization, requires the sample surface to be even/flat and sliced into thin sections (c. 10 µm). Furthermore, sample preparation steps can alter the analyte composition of the sample. The liquid microjunction-surface sampling probe (LMJ-SSP) is a robust sampling interface that enables surface profiling with minimal sample preparation. In conjunction with a conductance feedback system, the LMJ-SSP can be used to automatically sample uneven specimens. METHODS: A sampling stage was built with a modified 3D printer where the LMJ-SSP is attached to the printing head. This setup can scan across flat and even surfaces in a predefined pattern ("static sampling mode"). Uneven samples are automatically probed in "conductance sampling mode" where an electric potential is applied and measured at the probe. When the probe contacts the electrically grounded sample, the potential at the probe drops, which is used as a feedback signal to determine the optimal position of the probe for sampling each location. RESULTS: The applicability of the probe/sensing system was demonstrated by first examining the strawberry tissue using the "static sampling mode." Second, porcine tissue samples were profiled using the "conductance sampling mode." With minimal sample preparation, an area of 11 × 15 mm was profiled in less than 2 h. From the obtained results, adipose areas could be distinguished from non-adipose parts. The versatility of the approach was further demonstrated by directly sampling the bacteria colonies on agar and resected human kidney (intratumoral hemorrhage) specimens with thicknesses ranging from 1 to 4 mm. CONCLUSION: The LMJ-SSP in conjunction with a conductive feedback system is a powerful tool that allows for fast, reproducible, and automated assessment of uneven surfaces with minimal sample preparation. This setup could be used for perioperative assessment of tissue samples, food screening, and natural product discovery, among others.

Hyperspectral Visualization-Based Mass Spectrometry Imaging by LMJ-SSP: A Novel Strategy for Rapid Natural Product Profiling in Bacteria.

Mass spectrometry imaging (MSI) has been widely used to discover natural products (NPs) from underexplored microbiological sources. However, the technique is limited by incompatibility with complicated/uneven surface topography and labor-intensive sample preparation, as well as lengthy compound profiling procedures. Here, liquid micro-junction surface sampling probe (LMJ-SSP)-based MSI is used for rapid profiling of natural products from Gram-negative marine bacteria Pseudoalteromonas on nutrient agar media without any sample preparation. A conductance-based autosampling platform with 1 mm spatial resolution and an innovative multivariant analysis-driven method was used to create one hyperspectral image for the sampling area. NP discovery requires general spatial correlation between m/z and colony location but not highly precise spatial resolution. The hyperspectral image was used to annotate different m/z by straightforward color differences without the need to directly interrogate the spectra. To demonstrate the utility of our approach, the rapid analysis of Pseudoalteromonas rubra DSM6842, Pseudoalteromonas tunicata DSM14096, Pseudoalteromonas piscicida JCM20779, and Pseudoalteromonas elyakovii ATCC700519 cultures was directly performed on Agar. Various natural products, including prodiginine and tambjamine analogues, were quickly identified from the hyperspectral image, and the dynamic extracellular environment was shown with compound heatmaps. Hyperspectral visualization-based MSI is an efficient and sensitive strategy for direct and rapid natural product profiling from different Pseudoalteromonas strains.

Discontinuously Dewetting Solvent Arrays: Droplet Formation and Poly-Synchronous Surface Extraction for Mass Spectrometry Imaging Applications.

We describe a new liquid tissue stamping method called poly-synchronous surface extraction (PSSE) that utilizes an omniphobic substrate patterned with hydrophilic surface energy traps (SETs), which when wet with a solvent form a dense microdroplet array. When contacted with a tissue sample, each droplet locally extracts analytes from the tissue surface, which subsequentially can be analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-IMS) or ambient ionization-MS techniques. Optimization of the patterned surface with six different solvents was carried out to increase the droplet density, height, and reproducibility of volume deposition. Once optimized, sister slices of a strawberry (Fragaria × ananassa) were spatially extracted using the PSSE technique and the chemical distribution of selected compounds was analyzed with both MALDI-IMS and a lower resolution but faster ambient liquid microjunction surface sampling probe (LMJ-SSP) approach. Heat maps for target analytes for the PSSE approach are compared to those produced using traditional MALDI-IMS analysis. The PSSE method aligned well with direct analysis and demonstrated the potential to increase the speed of ambient MS tissue imaging techniques by decreasing the number of steps required for sample preparation.

Detection of Opioids on Mail/Packages Using Open Port Interface Mass Spectrometry (OPI-MS).

Opioids (and their more potent synthetic analogues) are used therapeutically as effective pain killers; however, recreational use and consequent overdoses are implicated in the deaths of thousands of people across the world annually. Trafficking of opioids and other illegal drugs through international mail has become a significant challenge for law enforcement personnel. Hundreds of millions of letters are sorted by the U.S. and Canadian postal services every day. Chemical analysis of this immense volume of mail requires a very fast sampling/detection method. This work explores the use of real-time mass spectrometry analysis with the recently developed Open Port Interface (OPI) for acoustically dispensed nanoliter volume sample droplets, a type of liquid microjunction surface sampling probe, for rapid and easy non-intrusive detection of fentanyl, heroin, and oxycodone. The OPI coupled to mass spectrometry is a novel sample introduction method that allows the rapid analysis of sample surfaces without preparation or modification. Opioids on different packaging materials (e.g., paper, bubble wrap, Ziploc bags) were rapidly (<10 s) interrogated by the OPI, and the sensitivities of the method compared. Furthermore, an opioid surrogate (caffeine) could be facilely detected on envelopes after processing through postal services.

Effects of electrospray mechanisms and structural relaxation on polylactide ion conformations in the gas phase: insights from ion mobility spectrometry and molecular dynamics simulations.

Recent advances in molecular dynamics (MD) simulations have made it possible to examine the behavior of large charged droplets that contain analytes such as proteins or polymers, thereby providing insights into electrospray ionization (ESI) mechanisms. In the present study, we use this approach to investigate the release of polylactide (PLA) ions from water/acetonitrile ESI droplets. We found that cationized gaseous PLA ions can be formed via various competing pathways. Some MD runs showed extrusion and subsequent separation of polymer chains from the droplet, as envisioned by the chain ejection model (CEM). On other occasions the PLA chains remained inside the droplets and were released after solvent evaporation to dryness, consistent with the charge residue model (CRM). Following their release from ESI droplets, the nascent gaseous PLA ions were subjected to structural relaxation for several µs in vacuo. The MD conformations generated in this way for various PLA charge states compared favorably to experimental results obtained by ion mobility spectrometry-mass spectrometry (IMS-MS). The structures of all PLA ions evolved during relaxation in the gas phase. However, some macroion species retained features that resembled their nascent structures. For this subset of ions, the IMS-MS response appears to be strongly correlated with the ESI release mechanism (CEM vs. CRM). The former favored extended structures, whereas the latter preferentially generated compact conformers.

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.

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.

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.

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.

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.

Collision-Induced Dissociation of Electrosprayed NaCl Clusters: Using Molecular Dynamics Simulations to Visualize Reaction Cascades in the Gas Phase.

Infusion of NaCl solutions into an electrospray ionization (ESI) source produces [Na(n+1)Cl n ]+ and other gaseous clusters. The n = 4, 13, 22 magic number species have cuboid ground state structures and exhibit elevated abundance in ESI mass spectra. Relatively few details are known regarding the mechanisms whereby these clusters undergo collision-induced dissociation (CID). The current study examines to what extent molecular dynamics (MD) simulations can be used to garner insights into the sequence of events taking place during CID. Experiments on singly charged clusters reveal that the loss of small neutrals is the dominant fragmentation pathway. MD simulations indicate that the clusters undergo extensive structural fluctuations prior to decomposition. Consistent with the experimentally observed behavior, most of the simulated dissociation events culminate in ejection of small neutrals ([NaCl] i , with i = 1, 2, 3). The MD data reveal that the prevalence of these dissociation channels is linked to the presence of short-lived intermediates where a relatively compact core structure carries a small [NaCl] i protrusion. The latter can separate from the parent cluster via cleavage of a single Na-Cl contact. Fragmentation events of this type are kinetically favored over other dissociation channels that would require the quasi-simultaneous rupture of multiple electrostatic contacts. The CID behavior of NaCl cluster ions bears interesting analogies to that of collisionally activated protein complexes. Overall, it appears that MD simulations represent a valuable tool for deciphering the dissociation of noncovalently bound systems in the gas phase. Graphical Abstract ᅟ.

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations.

Much remains to be learned about the way in which bound metal ions modulate the response of electrosprayed proteins and protein complexes to collisional excitation. Nonspecific metal adducts can affect the extent of collision-induced unfolding (CIU) and collision-induced dissociation (CID). Here, we examine how Na(+) and Ca(2+) adducts alter the CIU response of monomeric proteins under native electrospray conditions. Both of these metals are commonly encountered in biological samples. Measured collision cross sections are largely independent of metal adduction as long as in-source excitation is minimized. In contrast, under CIU conditions, the metal-adducted proteins are markedly more compact than their metal-free counterparts. This phenomenon is particularly pronounced for Ca(2+) binding, but Na(+) adducts have significant effects as well. Molecular dynamics simulations reproduce the experimentally observed trends. The simulations show that structural expansion of the collisionally unfolded proteins is limited by multidentate metal contacts that restrict the conformational freedom of the polypeptide chains. Multidentate interactions with carboxylates and other electron-rich moieties are to be anticipated for divalent metals such as Ca(2+). It is surprising that Na(+) also engages in multidentate ligation. Electrostatic mapping reveals that the propensity of both Na(+) and Ca(2+) to interact with multiple electron-rich groups is caused by ineffective charge shielding during ion pairing. Despite their compactness, the CIU structures of metalated proteins do not retain native-like elements. Instead, CIU generates inside-out conformations where previously surface-exposed hydrophilic side chains get buried along with most of the metal ions. Our findings caution that the observation of compact conformers after collisional excitation does not imply the survival of solution-like structural features. We also discuss possible implications of adduct-mediated effects for CIU fingerprinting studies.

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.

Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations.

The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here.

Exploring the mechanism of salt-induced signal suppression in protein electrospray mass spectrometry using experiments and molecular dynamics simulations.

Protein analyses by electrospray ionization (ESI) mass spectrometry can suffer from interferences caused by nonvolatile salts. The mechanistic basis of this effect remains to be fully investigated. In the current work we explore the behavior of proteins under native and denaturing conditions in the presence of NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl). All three salts interfere with the formation of "clean" [M + zH](z+) protein ions by progressively deteriorating spectral S/N ratios. We propose that salt interferences can be dissected into two independent aspects, i.e., (i) peak splitting by adduct formation and (ii) protein ion suppression. NaCl degrades the spectral quality by forming heterogeneous [M + zH + n(Na - H) + m(Cl + H)](z+) ions, while the integrated protein ion intensity remains surprisingly robust. Conversely, NBu4Cl does not cause any adduction, while dramatically reducing the protein ion yield. These findings demonstrate that adduct formation and protein ion suppression are indeed unrelated effects that may occur independently of one another. Other salts, such as CsCl, can give rise to a combination of the two scenarios. Molecular dynamics simulations of water droplets charged with either Na(+) or NBu4(+) provide insights into the mechanism underlying the observed effects. Na(+) containing droplets evolve relatively close to the Rayleigh limit (z/z(R) ≈ 0.74), whereas the z/z(R) values of NBu4(+) charged droplets are considerably lower (∼0.59). This difference is due to the high surface affinity of NBu4(+), which facilitates charge ejection from the droplet. We propose that the low z/z(R) values encountered in the presence of NBu4(+) suppress the Rayleigh fission of parent droplets in the ESI plume, thereby reducing the yield of progeny droplets that represent the precursors of gaseous protein ions. In addition, the rate of solvent evaporation is reduced in the presence of NBu4(+). Both of these factors lower the protein signal intensity. NaCl does not interfere with droplet fission, such that protein ions continue to form with high yield­albeit in heavily adducted form. Our findings expand on earlier proposals of charge competition as a key factor during the ESI process for salt-contaminated solutions.

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.