Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

Proton transfer reactions of ammonia, dimemylamine, diethylamine, and trimethylarnine with multiply protonated proteins generated by electrospray ionization (ESI) were examined to probe the relationship between solution and gas-phase protein structure and the relationship with ion-molecule reactivity. The ion-molecule reactions were carried out in an atmospheric pressure capillary inlet/reactor based upon an ESI interface to a quadrupole mass spectrometer. Two types of systems were explored: (1) proteins possessing cysteine-cysteine disulfide bonds and the analogous disulfide-reduced proteins, and (2) proteins sprayed from solution compositions where the protein has different conformations. While the cysteine-cysteine disulfide-bound proteins were more reactive than equally charged disulfide-reduced proteins under these conditions, no significant reactivity differences were noted for ions arising from different solution conformations. The effect of inlet/reactor temperature on charge distributions with and without amine reagent was also explored, demonstrating that thermal denaturation of proteins can occur in heated capillary inlets. The results are discussed in the context of recent results indicating the persistence of at least some higher order protein structure in the gas phase.     

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer

Proton transfer reaction of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between “native” and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivity to an ion's three-dimensional structure or “compactness.”     

On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization

A relatively simple model for calculation of the energetics of gas-phase proton transfer reactions and the maximum charge state of multiply protonated ions formed by electrospray ionization is presented. This model is based on estimates of the intrinsic proton transfer reactivity of sites of protonation and point charge Coulomb interactions. From this model, apparent gas-phase basicities (GB^app) of multiply protonated ions are calculated. Comparison of this value to the gas-phase basicity of the solvent from which an ion is formed enables a maximum charge state to be calculated. For 13 commonly electrosprayed proteins, our calculated maximum charge states are within an average of 6% of the experimental values reported in the literature. This indicates that the maximum charge state for proteins is determined by their gas-phase reactivity. Similar results are observed for peptides with many basic residues. For peptides with few basic residues, we find that the maximum charge state is better correlated to the charge state in solution. For low charge state ions, we find that the most basic sites Arg, Lys, and His are preferentially protonated. A significant fraction of the less basic residues Pro, Trp, and Gln are protonated in high charge state ions. The calculated GB^app of individual protonation sites varies dramatically in the high charge state ions. From these values, we calculate a reduced cross section for proton transfer reactivity that is significantly lower than the Langevin collision frequency when the GB^app of the ion is approximately equal to the GB of the neutral base.     

Modeling the maximum charge state of arginine-containing peptide ions formed by electrospray ionization

A model for the gas-phase proton transfer reactivity of multiply protonated molecules is used to quantitatively account for the maximum charge states of a series of arginine-containing peptide ions measured by Downard and Biemann (Int. J. Mass Spectrom. Ion Processes 1995, 148, 191-202). We find that our calculations account exactly for the maximum charge state for 7 of the 10 peptides and are off by one charge for the remaining 3. These calculations clearly predict the trend in maximum charge states for these peptides and provide further evidence that the maximum charge state of ions formed by electrospray ionization is determined by their gas-phase proton transfer reactivity.     

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Effects of Select Anions from the Hofmeister Series on the Gas-Phase Conformations of Protein Ions Measured with Traveling-Wave Ion Mobility Spectrometry/Mass Spectrometry

The gas-phase conformations of ubiquitin, cytochrome c, lysozyme, and α-lactalbumin ions, formed by electrospray ionization (ESI) from aqueous solutions containing 5 mM ammonium perchlorate, ammonium iodide, ammonium sulfate, ammonium chloride, ammonium thiocyanate, or guanidinium chloride, are examined using traveling-wave ion mobility spectrometry (TWIMS) coupled to time-of-flight (TOF) mass spectrometry (MS). For ubiquitin, cytochrome c, and α-lactalbumin, adduction of multiple acid molecules results in no significant conformational changes to the highest and lowest charge states formed from aqueous solutions, whereas the intermediate charge states become more compact. The transition to more compact conformers for the intermediate charge states occurs with fewer bound H₂SO₄ molecules than HClO₄ or HI molecules, suggesting ion-ion or salt-bridge interactions are stabilizing more compact forms of the gaseous protein. However, the drift time distributions for protein ions of the same net charge with the highest levels of adduction of each acid are comparable, indicating that these protein ions all adopt similarly compact conformations or families of conformers. No significant change in conformation is observed upon the adduction of multiple acid molecules to charge states of lysozyme. These results show that the attachment of HClO₄, HI, or H₂SO₄ to multiply protonated proteins can induce compact conformations in the resulting gas-phase protein ions. In contrast, differing Hofmeister effects are observed for the corresponding anions in solution at higher concentrations.     

Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers

The origin of asymmetric charge and mass partitioning observed for gas-phase dissociation of multiply charged macromolecular complexes has been hotly debated. These experiments hold the potential to provide detailed information about the interactions between the macromolecules within the complex. Here, this unusual phenomenon of asymmetric charge partitioning is investigated for several protein homodimers. Asymmetric charge partitioning in these ions depends on a number of factors, including the internal energy, charge state, and gas-phase conformation of the complex, as well as the conformational flexibility of the protein monomer in the complex. High charge states of both cytochrome c and disulfide-reduced alpha-lactalbumin homodimers dissociate by a symmetrical charge partitioning process in which both fragment monomers carry away roughly an equal number of charges. In contrast, highly asymmetric charge partitioning dominates for the lower charge states. Cytochrome c dimer ions with eleven charges formed by electrospray ionization from two solutions in which the solution-phase conformation differs dissociate with dramatically different charge partitioning. These results demonstrate that these gas-phase complexes retain a clear "memory" of the solution from which they are formed, and that information about their solution-phase conformation can be obtained from these gas-phase dissociation experiments. Cytochrome c dimer ions formed from solutions in which the conformation of the protein is native show greater asymmetric charge partitioning with increasing ion internal energy. Cytochrome c dimers that are conformationally constrained with intramolecular cross-linkers undergo predominantly symmetric charge partitioning under conditions where asymmetric charge partitioning is observed for cytochrome c dimers without cross-links. Similar results are observed for alpha-lactalbumin homodimers. These results provide convincing evidence that the origin of asymmetric charge partitioning in these homodimers is the result of one of the protein monomers unfolding in the dissociation transition state. A mechanism that accounts for these observations is proposed.     

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.     

Decharging of globular proteins and protein complexes in electrospray

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.     

Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes

The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH(3)Ac, Et(2)NH(2)Ac and Et(3)NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH(3), EtNH(2), Et(2)NH and Et(3)N. Charge states derived from evaluated apparent gas-phase basicities GB(app) of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H(3)O(+) ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein-complex equilibrium in aqueous solution.     

Determination of gas phase protein ion densities via ion mobility analysis with charge reduction

We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1?:?49.5?:?49.5, formic acid?:?methanol?:?water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.     

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.     

Defect formation on surfaces bombarded by energetic multiply charged proteins: Implications for the conformation of gas-phase electrosprayed ions

Indirect information on the conformation of highly charged molecular ions may be obtained by monitoring their collisional cross sections and the course of simple gas-phase reactions such as hydrogen-deuterium exchange. In this work, another indirect but more visually oriented approach is explored: electrosprayed protein ions are accelerated toward a highly oriented pyrolytic graphite surface and the resulting single-ion defects are imaged by scanning force and tunneling microscopy. All protein impacts generated shallow hillocks: the shapes depended on the identity and charge state of the incident protein. Lysozyme and myoglobin, both compact, globular proteins in the native state, produced compact, almost circular hillocks. However, hillocks generated by myoglobin that had been denatured in the solution phase were elongated, and the elongation was positively correlated with the charge state of the ion. It appears that structural information about gas-phase multiply charged proteins can be derived from imprints generated by energetic protein impacts on surfaces.     

Apparent gas-phase acidities of multiply protonated peptide ions: Ubiquitin,insulin B,and renin substrate

The gas-phase deprotonation reactions of multiply protonated bovine ubiquitin, insulin chain B, and renin substrate tetradecapeptide ions have been studied in a Fourier transform ion cyclotron resonance mass spectrometer coupled with an external electrospray source. Rate constants were measured for the reactions of these peptide ions with a series of reference compounds of known gas-phase basicities ranging from 195.6 to 232.6 kcal/mol. The apparent gas-phase acidities (GA_app) of the multiply protonated peptide ions [M + nH]ⁿ⁺ were determined with deprotonation reactions. The deduced values of GA_app show a strong dependence on the charge states of the multiply protonated peptide ions. In general, the values decrease as the charge states of the peptide ions increase. For ubiquitin ions, the determined GA_apps values decrease from> 232.6 to 205.0 kcal/mol for n = 4-13; for insulin B ions, the GA_apps decrease from> 232.6 to 198.2 kcal/mol for n = 2-5; for renin substrate ions, the GA_apps decrease from 221.6 to < 195.6 kcal/mol for n = 2-4. Interestingly, at a given mass-to-charge ratio, the GA_apps of these peptide ions agree within 10 kcal/mol despite large differences in their mass and charge. The ubiquitin and insulin B ions generated under the present conditions reveal multiple isomers at certain charge states,n = 4, 5, 6, 12 for ubiquitin and n = 4, 5 for insulin B, as evidenced by the fact that the isomers display distinctively different deprotonation reaction rates with certain reference compounds.     

Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization

Electrospray ionization (ESI) of denatured proteins produces a broad distribution of multiply-charged ions leading to multiple peaks in the mass spectrum. We investigated changes in the positive-mode ESI charge state distribution produced by several chemical modifications of denatured proteins. Capping carboxylic acid groups with neutral functional groups yields little change in charge state distribution compared with unmodified proteins. The results indicate that carboxyl groups do not play a significant role in the positive charging of denatured proteins in ESI. The modification of proteins with additional basic sites or fixed positive charges generates substantially higher charge states, providing evidence that the number of ionizable sites, rather than molecular size and shape, determines ESI charging for denatured proteins. Fixed charge modification also significantly reduces the number of protons acquired by a protein, in that the charge state envelope is not increased by the full number of fixed charges appended. This result demonstrates that Coulombic repulsion between positive charges plays a significant role in determining charge state distribution by affecting the gas-phase basicity of ionizable sites. Addition of fixed-charge moieties to a protein is a useful approach for shifting protein charge state distributions to higher charge states, and with further work, it may help limit the distribution of protein ions to fewer charge states.     

ESIprot: a universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data

Electrospray ionization (ESI) ion trap mass spectrometers with relatively low resolution are frequently used for the analysis of natural products and peptides. Although ESI spectra of multiply charged protein molecules also can be measured on this type of devices, only average spectra are produced for the majority of naturally occurring proteins. Evaluating such ESI protein spectra would provide valuable information about the native state of investigated proteins. However, no suitable and freely available software could be found which allows the charge state determination and molecular weight calculation of single proteins from average ESI‐MS data. Therefore, an algorithm based on standard deviation optimization (scatter minimization) was implemented for the analysis of protein ESI‐MS data. The resulting software ESIprot was tested with ESI‐MS data of six intact reference proteins between 12.4 and 66.7 kDa. In all cases, the correct charge states could be determined. The obtained absolute mass errors were in a range between ?0.2 and 1.2 Da, the relative errors below 30 ppm. The possible mass accuracy allows for valid conclusions about the actual condition of proteins. Moreover, the ESIprot algorithm demonstrates an extraordinary robustness and allows spectral interpretation from as little as two peaks, given sufficient quality of the provided m/z data, without the necessity for peak intensity data. ESIprot is independent from the raw data format and the computer platform, making it a versatile tool for mass spectrometrists. The program code was released under the open‐source GPLv3 license to support future developments of mass spectrometry software. Copyright © 2010 John Wiley & Sons, Ltd.     

Proton transfer reactions of derivatized fullerene trications

The occurrence or absence of proton transfer from derivatized fullerene trications C₆₀H³⁺, to the parent neutral XH, is employed to obtain upper or lower limits to the apparent gas-phase acidity GA_app as well as to the estimated absolute gas-phase acidity. A comparison with the reactivity of analogous dicationic adducts indicates that C₆₀XH³⁺ is generally more acidic than C₆₀XH²⁺: for example, the difference in GA values for C₆₀NCCH ₃ ⁿ⁺ (n 5 2, 3; formed in the addition reaction of C ₆₀ ⁿ⁺ with CH₃CN) is estimated to be at least 42 kcal mol21. In almost all instances, estimated GA values for the tricationic adducts are far below the gas-phase basicities of the parent neutral XH, mirroring a trend seen from the proton-transfer reactivity of multiply protonated protein molecules: a qualitative difference between the fullerene adducts and the multiply protonated biomolecules routinely produced by electrospray ionization is that the fullerene adduct ions (with the exception of C₆₀NH ₃ ³⁺ ) appear to possess only one acidic proton.     

Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools

This tutorial provides an overview of the evolution of some of the key concepts in the gas-phase fragmentation of different classes of peptide ions under various conditions [e.g. collision-induced dissociation (CID) and electron transfer dissociation (ETD)], and then demonstrates how these concepts can be used to develop new methods. For example, an understanding of the role of the mobile proton and neighboring group interactions in the fragmentation reactions of protonated peptides has led to the design of the 'SELECT' method. For ETD, a model based on the Landau-Zener theory reveals the role of both thermodynamic and geometric effects in the electron transfer from polyatomic reagent anions to multiply protonated peptides, and this predictive model has facilitated the design of a new strategy to form ETD reagent anions from precursors generated via ESI. Finally, two promising, emerging areas of gas-phase ion chemistry of peptides are also described: (1) the design of new gas-phase radical chemistry to probe peptide structure, and (2) selective cleavage of disulfide bonds of peptides in the gas phase via various physicochemical approaches.     

Ion trap collisional activation of disulfide linkage intact and reduced multiply protonated polypeptides.

The presence of disulfide linkages in multiply charged polypeptide ions tends to inhibit the formation of structurally informative product ions under conventional quadrupole ion trap collisional activation conditions. In particular, fragmentation that requires two cleavages (i.e., cleavage of a disulfide linkage and a peptide linkage) is strongly suppressed. Reduction of the disulfide linkage(s) by use of dithiothreitol yields parent ions upon electrospray without this complication. Far richer structural information is revealed by ion trap collisional activation of the disulfide-reduced species than from the native species. These observations are illustrated with doubly protonated native and reduced somatosin, the [M + 5H](5+) ion of native bovine insulin and the [M + 4H](4+) and [M + 3H](3+) ions of the B-chain of bovine insulin produced by reduction of the disulfide linkages in insulin, and the [M + 11H](11+) ion of native chicken lysozyme and the [M + 11H](11+) and [M + 14H](14+) ions of reduced lysozyme. In each case, the product ions produced by ion trap collisional activation were subjected to ion/ion proton transfer reactions to facilitate interpretation of the product ion spectra. These studies clearly suggest that the identification of polypeptides with one or more disulfide linkages via application of ion trap collisional activation to the multiply charged parent ions formed directly by electrospray could be problematic. Means for cleaving the disulfide linkage, such as reduction by dithiothreitol prior to electrospray, are therefore desirable in these cases.     

Charge carrier field emission determines the number of charges on native state proteins in electrospray ionization

Although multiple charging in electrospray ionization (ESI) is essential to protein mass spectrometry, the underlying mechanism of multiple charging has not been explicated. Here, we present a new theory to describe ESI of native-state proteins and predict the number of excess charges on proteins in ESI. The theory proposes that proteins are ionized as charged residues in ESI, as they retain residual excess charges after solvent evaporation and do not desorb from charged ESI droplets. However, their charge state is not determined by the Rayleigh limit of a droplet of similar size to the protein; rather, their final charge state is determined by the electric field-induced emission of small charged solute ions and clusters from protein-containing ESI droplets. This theory predicts that the number of charges on a protein in ESI should be directly proportional to the square of the gas-phase protein diameter and to E*, the critical electric field strength at which ion emission from droplets occurs. This critical field strength is determined by the properties of the excess charge carriers (i.e., the solute) in droplets. Charge-state measurements of native-state proteins with molecular masses in the 5-76 kDa range in ammonium acetate and triethylammonium bicarbonate are in excellent agreement with theoretical predictions and strongly support the mechanism of protein ESI proposed here.