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 structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The ion atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.     

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.     

A new approach for the study of gas-phase ion-ion reactions using electrospray ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)₃) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]ⁿ⁺ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.     

Coulomb effects in binding of heme in gas-phase ions of myoglobin

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry. With negative ions, neutral heme is lost regardless of the charge state of Fe in solution. It is likely that the Fe(3+) is reduced to Fe(2+) in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed with ions formed from ferromyoglobin in solution, and positive heme loss with ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model potential with a long-range Coulomb repulsion and short-range attraction between the heme and protein.     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

Applying charge discrimination with electrospray ionization—mass spectrometry to protein analyses

Electrospray ionization with a magnetic sector mass spectrometer and scanning array detector has unique advantages for sensitive analyses of large biomolecules. The ability to discriminate against low charge state ions (smaller peptides, buffers and salts, background ions) allows for detection of more highly charged ions from proteins present at much lower concentration relative to the small ions from buffers and detergents present. Low femtomole detection limits can be achieved for proteins greater than 100 ku. The charge discrimination phenomenon is more pronounced for higher charged ions, and especially for large biomolecules. Although the charge distribution for the monomer (66 ku) and dimer (133 ku) species of bovine serum albumin overlap, both species can be ascertained readily in a mixture because the lower charged monomer ions have higher optimum microchannel plate voltages than the higher charged dimer ions. Protein-containing solutions can be analyzed directly by electrospray ionization—mass spectrometry (ESI-MS) with array detection, which eliminates time-consuming separation and sample cleanup procedures. For example, heme-containing proteins can be directly detected from ESI-MS of human blood (hemoglobin) as well as from raw meat juices (hemoglobin and myoglobin).     

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of alpha-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.     

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.     

Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, ‘native-like’ geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.     

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions

Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and β-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM′ (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.     

Charge propagation in "ion channel sensors" based on protein-modified electrodes and redox marker ions

The mechanism of charge propagation in "ion channel sensors" (ICSs) consisting of gold electrodes modified with a layer of charged proteins and highly charged redox-active marker ions in solution was investigated by electrochemical techniques, QCM and AFM. The study is based on seven proteins (concanavalin A, cytochrome c, glucose oxidase, lysozyme, thyroglobulin, catalase, aldolase, and EF1-ATPase) in combination with seven electroactive marker ions ([Fe(CN)6]3-, [Fe(CN)6]4-, [Ru(NH3)6]3+, mono-, di-, and trimeric viologens), as well as a series of suppressor and enhancer ions leading to the following general statements: (i) electrostatic binding of charged marker ions to the domains of the protein is a prerequisite for an electrochemical current and (ii) charge propagation through the layer consists of electron hopping along surface-confined marker ions into the pores between adsorbed proteins. It is further shown that (iii) marker ions and suppressor ions with identical charge compete for oppositely charged sites on the protein domain, (iv) electrostatically bound multilayers of marker or enhancer ions with alternating charge form on a charged protein domain, and (v) self-exchange and exergonic ET catalysis between adsorbed marker ions and marker ions in solution take place. In addition to fundamental insight into the mechanism of charge propagation, valuable information for the design, optimization, and tailoring of new biosensors based on the ICS concept is demonstrated by the current findings.     

FT-ICRMS distinguishes the mechanism of the charge state reduction for multiply charged protein cations admixed with redox reagents in ESI

Recently, we reported on a phenomenon in which multiply charged protein cations produced by electrospray ionization could be reduced to lower and narrower charge state distributions when admixed with reducing reagents 1,4-benzoquinone or quinhydrone. Circular dichroism spectra of the proteins indicated that secondary and tertiary structural changes upon addition of these reducing reagents were negligible, thus eliminating conformational effects as playing a role in the charge reduction mechanism. Furthermore, the extent of charge state reduction did not correspond with gas-phase basicities of the redox reagents, suggesting that solution-phase, and not gas-phase, behavior dominates the observed charge state reduction. The relatively low resolution of the triple quadrupole employed did not make it possible to distinguish isotopic distributions of the multiply charged cations in order to determine whether the observed phenomenon was the result of proton-transfer reactions between the multiply charged cations and the reducing reagent or because of electron transfer from the reducing reagent to the protein cations. Here, high-resolution ESI-Fourier transform ion cyclotron resonance mass spectrometry of several peptide amides in the presence of a redox reagent show isotopic distributions that are consistent only with the proton-transfer mechanism.     

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK_a or pK_b of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry.     

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.     

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.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

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.     

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB(app)) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GB(app) values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom.2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is proposed.