Protein Charge State Research Articles

Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization.

Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations. To address this limitation, we combined UVPD-MS with drift tube ion mobility, offering potential to acquire conformationally selective MS/MS information. A low-pressure drift tube (LPDT) Orbitrap mass spectrometer equipped with 193 nm UVPD capabilities enables the analysis of protein conformers through the analysis of arrival time distributions (ATDs) of individual fragment ions. ATDs of fragment ions are compared for different backbone cleavage sites of the protein or different precursor charge states to give information about regions of potential folding or elongation. This integrated platform offers promise for advancing our understanding of protein structures in the gas phase.

Conformational and Structural Characterization of Knotted Proteins.

Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 52 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 61 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature (T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å2 and ∼3250-15,385 Å2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry-mass spectrometry.

Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.

Modeling pH-Dependent Biomolecular Photochemistry.

The tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to the photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics using a combination of constant-pH molecular dynamics and semiclassical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena sensory rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties, such as the isomerization quantum yield or decay rates. We decompose our results into single-titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy.

Effects of Amino Acid Additives on Protein Stability during Electrothermal Supercharging in ESI-MS.

The surprising formation of highly charged protein ions from aqueous ammonium bicarbonate solution is a fascinating phenomenon referred to as electrothermal supercharging (ETS). Although the precise mechanism involved is not clearly understood, previous studies predominantly suggest that ETS is due to native protein destabilization in the presence of bicarbonate anion inside the electrospray ionization droplets under high temperatures and spray voltages. To evaluate existing hypotheses surrounding the underlying mechanism of ETS, the effects of several additives on protein charging under ETS conditions were investigated. The changes in the protein charge state distributions were compared by measuring the ratios between the intensities of highest intensity charge states of native and unfolded protein envelopes and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important role in ETS compared to spray voltage, especially when using a nebulized microelectrospray ionization source. Moreover, the effect of amino acids on ETS were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by these additives in bulk solution. Among the natural amino acids, protein supercharging was significantly reduced by proline and glycine; however, imidazole provided the highest degree of noncovalent complex stabilization against ETS, outperforming the amino acids. Overall, our study shows that the simple addition of stabilizing reagents such as proline and imidazole can reduce the extent of apparent protein unfolding and supercharging in ammonium bicarbonate solution and provide evidence against the roles of charge depletion and thermal unfolding during ETS.

Probing Acid-Induced Compaction of Denatured Proteins by High-Pressure Electrospray Mass Spectrometry.

Further increase in the acidity in the most denaturing acidic solution is known to induce compaction of the fully unfolded protein into a compact molten globule. The phenomenon of "acid-induced folding of proteins" takes place at pH ∼1 in strong acid aqueous solutions with high electrical conductivity and surface tension, a condition that is difficult to handle using conventional electrospray ionization methods for mass spectrometry. Here, high-pressure electrospray ionization (HP-ESI) is used to produce well-resolved mass spectra for proteins in strong acids with pH as low as 1. The compaction of protein conformation is indicated by a large shift in the charge state from high charges to native-like low charges. The addition of salt to the protein in the most denaturing condition also reproduces the compaction effect, thereby supporting the role of anions in this phenomenon. Similar compaction of proteins is also observed in organic solvent/acid mixtures. The charge state of the compacted protein depends on the type of anions that formed ion pairs with a positive charge on the protein. The dissociation of ion pairs during the ionization process forms neutral acids that can be observed by HP-ESI using a soft ion introduction configuration.

Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes.

Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures. Here, we discuss the first application of our computational structure relaxation approximation [Bleiholder, C.; et al. J. Phys. Chem. B 2019, 123 (13), 2756-2769] to assign structures of protein complexes in the range from ∼16 to ∼60 kDa from their "native" IM/MS spectra. Our analysis shows that the computed IM/MS spectra agree with the experimental spectra within the errors of the methods. The structure relaxation approximation (SRA) indicates that native backbone contacts appear largely retained in the absence of solvent for the investigated protein complexes and charge states. Native contacts between polypeptide chains of the protein complex appear to be retained to a comparable extent as contacts within a folded polypeptide chain. Our computations also indicate that the hallmark "compaction" often observed for protein systems in native IM/MS measurements appears to be a poor indicator of the extent to which native residue-residue interactions are lost in the absence of solvent. Further, the SRA indicates that structural reorganization of the protein systems in IM/MS measurements appears driven largely by remodeling of the protein surface that increases its hydrophobic content by approximately 10%. For the systems studied here, this remodeling of the protein surface appears to occur mainly by structural reorganization of surface-associated hydrophilic amino acid residues not associated with β-strand secondary structure elements. Properties related to the internal protein structure, as assessed by void volume or packing density, appear unaffected by remodeling of the surface. Taken together, the structural reorganization of the protein surface appears to be generic in nature and to sufficiently stabilize protein structures to render them metastable on the time scale of IM/MS measurements.

Effects of charge on protein ion structure: Lessons from cation-to-anion, proton-transfer reactions.

Collision cross-section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross-section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation-to-anion, proton-transfer reactions (CAPTR), a method for generating a series of charge-reduced protein cations by reacting quadrupole-selected cations with even-electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge-manipulation strategies and review the results of various CAPTR-based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

A Subtle Change in Nanoflow Rate Alters the Ionization Response As Revealed by Scanning Voltage ESI-MS.

The small charged droplet generated from the nanoelectrospray ionization (nanoESI) source at nL/min flow rate gives its unique feature of high-performance ionization. A continuous scan of the flow rate in this regime can trace the effect of droplet size in greater detail for a better understanding of the ionization process. To date, such practical implementation is hindered by the lack of a suitable liquid pump and the reproducibility of microcapillaries-based systems. Here, offline nanoESI mass spectrometry with a continuously varying flow rate in a dynamic range of several hundred pL/min to ∼100 nL/min was performed by the precision scanning of ESI high voltage (HV). The principle is based on the new paradigm of generating nanoelectrospray from a large Taylor cone with a known spray current-flow rate relationship. The instantaneous flow rate controlled by the HV was determined by simultaneous measurement of the spray current. The system is successfully applied to reveal the role of nanoflow rate on the average charge state of proteins, analysis of analyte mixture, and desalting effect. With the use of a buffer solution with high electric conductivity, a highly controllable oxidative modification was also observed by tuning the flow rate below a threshold of ∼5 nL/min, a finding that has potential application to on-demand oxygen labeling.

Effective Amino Acid Sequencing of Intact Filgrastim by Multimodal Mass Spectrometry with Topdownr.

Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N- and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.

Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Effect of protein adsorption on the dissolution kinetics of silica nanoparticles

Nanoparticulate systems in the presence of proteins are highly relevant for various biomedical applications such as photo-thermal therapy and targeted drug delivery. These involve a complex interplay between the charge state of nanoparticles and protein, the resulting protein conformation, adsorption equilibrium and adsorption kinetics, as well as particle dissolution. SiO2 is a common constituent of bioactive glasses used in biomedical applications. In this context, the dissolution behavior of silica particles in the presence of a model protein, bovine serum albumin (BSA), at physiologically relevant pH conditions was studied. Sedimentation analysis using an analytical ultracentrifuge showed that BSA in the supernatant solution is not affected by the presence of silica nanoparticles. However, zeta potential measurements revealed that the presence of the protein alters the particles’ charge state. Adsorption and dissolution studies demonstrated that the presence of the protein significantly enhances the dissolution kinetics via interactions of positively charged amino acids in the protein with the negative silica surface and interaction of BSA with dissolved silicate species. Our study provides comprehensive insights into the complex interactions between proteins and oxide nanoparticles and establishes a reliable protocol paving the way for future investigations in more complex systems involving biological solutions as well as bioactive materials.

Ca2+/Sr2+ Selectivity in Calcium-Sensing Receptor (CaSR): Implications for Strontium's Anti-Osteoporosis Effect.

The extracellular calcium-sensing receptor (CaSR) controls vital bone cell functions such as cell growth, differentiation and apoptosis. The binding of the native agonist (Ca2+) to CaSR activates the receptor, which undergoes structural changes that trigger a cascade of events along the cellular signaling pathways. Strontium (in the form of soluble salts) has been found to also be a CaSR agonist. The activation of the receptor by Sr2+ is considered to be the major mechanism through which strontium exerts its anti-osteoporosis effect, mostly in postmenopausal women. Strontium-activated CaSR initiates a series of signal transduction events resulting in both osteoclast apoptosis and osteoblast differentiation, thus strengthening the bone tissue. The intimate mechanism of Sr2+ activation of CaSR is still enigmatic. Herewith, by employing a combination of density functional theory (DFT) calculations and polarizable continuum model (PCM) computations, we have found that the Ca2+ binding sites 1, 3, and 4 in the activated CaSR, although possessing a different number and type of protein ligands, overall structure and charge state, are all selective for Ca2+ over Sr2+. The three binding sites, regardless of their structural differences, exhibit almost equal metal selectivity if they are flexible and have no geometrical constraints on the incoming Sr2+. In contrast to Ca2+ and Sr2+, Mg2+ constructs, when allowed to fully relax during the optimization process, adopt their stringent six-coordinated octahedral structure at the expense of detaching a one-backbone carbonyl ligand and shifting it to the second coordination layer of the metal. The binding of Mg2+ and Sr2+ to a rigid/inflexible calcium-designed binding pocket requires an additional energy penalty for the binding ion; however, the price for doing so (to be paid by Sr2+) is much less than that of Mg2+. The results obtained delineate the key factors controlling the competition between metal cations for the receptor and shed light on some aspects of strontium’s therapeutic effects.

Advanced Strategies for Proton-Transfer Reactions Coupled with Parallel Ion Parking on a 21 T FT-ICR MS for Intact Protein Analysis.

Proton-transfer reactions (PTRs) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to "concentrate", in the gas phase, nearly all the signals from an intact protein charge state envelope into a single charge state, improving the signal-to-noise ratio (S/N) by 10× or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides a tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call "PTR-MS1-PIP", generates a pseudo-MS1 spectrum derived from ions that are exposed to the PTR reagent and PIP waveforms but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in the spectral S/N of intact proteins. This permits the observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the PTR rate without compromise to the parking efficiency. We demonstrate that this process, called "rapid park", can improve reaction rates by 5-10× and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS1 and rapid park provides a 10-fold reduction in ion injection time, improving the rate of tandem MS sequencing.

Effects of non-ionic and zwitterionic detergents on soluble proteins during native mass spectrometry experiments

Detergents are commonly employed for solubilization and stabilization of integral membrane proteins. Their effects on the charge states of detergent-solubilized membrane proteins in native mass spectrometry measurements have previously been described. These effects can be mediated through the transmembrane region directly interacting with the detergent micelle or through the soluble domains of the proteins getting in close contact with detergent micelles during electrospray ionization. Here, we explore the effects of detergent micelles on soluble proteins during native mass spectrometry aiming at distinguishing these two events. Specifically, we employ two non-ionic and one zwitterionic detergents as well as a variety of standard proteins differing in size, oligomeric state and surface hydrophobicity, and evaluate the observed charge states in the absence and presence of detergents. Using ion mobility-mass spectrometry, we assess the proteins' stability as well as the ability to maintain non-covalent interactions with ligands. Finally, we examine lipid transfer from mixed detergent-lipid micelles containing the various detergents to soluble proteins. In summary, we found that C8E4 detergent reduces the proteins’ charge states, stabilizes the proteins and facilitates lipid transfer.

Relation between Ejection Mechanism and Ion Abundance in the Electric Double Layer of Droplets.

Charged droplets have been associated with distinct chemical reactivity. It is assumed that the composition of the surface layer plays a critical role in enhancing the reaction rates in the droplets relative to their bulk solution counterparts. We use atomistic modeling to relate the localization of ions in the surface layer to their ejection propensity. We find that ion ejection takes place via a two-stage process. First, a conical protrusion emerges as a result of a global droplet deformation that is insensitive to the locations of the single ions. The ions are subsequently ejected as they enter the conical regions. The study provides mechanistic insight into the ion-evaporation mechanism, which can be used to revise the commonly used ion-evaporation models. We argue that atomistic molecular dynamics simulations of minute nanodrops do not sufficiently distinguish the ion-evaporation mechanism from a Rayleigh fission. We explain mass spectrometry data on the charge state of small globular proteins and the existence of supercharged droplet states that have been detected in experiments.

Facile Fabrication of Protein-Macrocycle Frameworks.

Precisely defined protein aggregates, as exemplified by crystals, have applications in functional materials. Consequently, engineered protein assembly is a rapidly growing field. Anionic calix[n]arenes are useful scaffolds that can mold to cationic proteins and induce oligomerization and assembly. Here, we describe protein-calixarene composites obtained via cocrystallization of commercially available sulfonato-calix[8]arene (sclx8) with the symmetric and “neutral” protein RSL. Cocrystallization occurred across a wide range of conditions and protein charge states, from pH 2.2–9.5, resulting in three crystal forms. Cationization of the protein surface at pH ∼ 4 drives calixarene complexation and yielded two types of porous frameworks with pore diameters >3 nm. Both types of framework provide evidence of protein encapsulation by the calixarene. Calixarene-masked proteins act as nodes within the frameworks, displaying octahedral-type coordination in one case. The other framework formed millimeter-scale crystals within hours, without the need for precipitants or specialized equipment. NMR experiments revealed macrocycle-modulated side chain pKa values and suggested a mechanism for pH-triggered assembly. The same low pH framework was generated at high pH with a permanently cationic arginine-enriched RSL variant. Finally, in addition to protein framework fabrication, sclx8 enables de novo structure determination.

Influence of protein ion charge state on 213 nm top-down UVPD.

Ultraviolet photodissociation (UVPD) is a powerful and rapidly developing method in top-down proteomics. Sequence coverages can exceed those obtained with collision- and electron-induced fragmentation methods. Because of the recent interest in UVPD, factors that influence protein fragmentation and sequence coverage are actively debated in the literature. Here, we performed top-down 213 nm UVPD experiments on a 7 T Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for the model proteins ubiquitin, myoglobin and cytochrome c that were electrosprayed from native, denaturing and supercharging solutions in order to investigate the effect of protein charge states on UVPD fragments. By performing UVPD in ultrahigh vacuum, factors associated with collisional cooling and any ion activation during transfer between mass analyzers can be largely eliminated. Sequence coverage increased from <10% for low charge states to >60% for high charge states for all three proteins. This trend is influenced by the overall charge state, i.e., charges per number of amino acid residues, and to a lesser degree by associated structural changes of protein ions of different charge states based on comparisons to published collision-cross section measurements. To rationalize this finding, and correlate sequence ion formation and identity with the number and location of protons, UVPD results were compared to protonation sites predicted based on electrostatic modelling. Assuming confined protonation sites, these results indicate the presence of two general fragmentation types; i.e., charge remote and charge directed. For moderately high protein charge states, fragment ions mostly originate in regions between likely protonation sites (charge remote), whereas sequence ions of highly charge protein ions occur either near backbone amide protonation sites at low-basicity residues (charge directed) or at charge remote sites (i.e., high-basicity residues). Overall, our results suggest that top-down 213 UVPD performance in the zero-pressure limit depends strongly on protein charge states and protonation sites can influence the location of backbone cleavages.

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.