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.     

Charged states of proteins. Reactions of doubly protonated alkyldiamines with NH(3): solvation or deprotonation. Extension of two proton cases to multiply protonated globular proteins observed in the gas phase

The apparent gas-phase basicities (GB(app)'s) of basic sites in multiply protonated molecules, such as proteins, can be approximately predicted. An approach used by Williams and co-workers was to develop an equation for a diprotonated system, NH(3)(CH(2))(7)NH(3)(2+), and then extend it with a summation of pairwise interactions to multiply protonated systems. Experimental determinations of the rates of deprotonation of NH(3)(CH(2))(7)NH(3)(2+) by a variety of bases B, in the present work, showed that GB(app) = GB(NH(3)) = 196 kcal/mol. This result is supported also by determinations of the equilibria: NH(3)(CH(2))(p)NH(3)(2+) + NH(3) = NH(3)(CH(2))(p)NH(3) x NH(3)(2+), for p = 7, 8, 10, 12. The described experimental GB(app) is 14 kcal/mol higher than the value predicted by the equation used by Williams and co-workers but in agreement with an ab initio result by Gronert. Equations based on electrostatics are developed for the two proton and multiproton systems which allow the evaluation of GB(app) of the basic sites on proteins. These are applied for the evaluation of GB(app) of the basic sites and of N(SB), the maximum number of protons that the nondenatured proteins, carbonic anhydrase (CAII), cytochrome c (CYC), and pepsin, can hold. The N(SB) values are compared with the observed charges, Z(obs)'s, when the nondenatured proteins are produced by electrospray and found in agreement with the proposal by de la Mora that Z(obs) is determined by the number of charges provided by the droplet that contains the protein, according to the charge residue model (CRM). The GB(app) values of proteins have many other applications. They can be compared with experimental measurements and are also needed for the understanding of the thermal denaturing of charged proteins and the thermal dissociation of charged protein complexes.     

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.     

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.     

胺丶醇丶醚类化合物气相碱性的CNDO/2计算

The gas-phase basicities of compounds can be measured by their proton affinities. In this paper we he calculated the gas-phase basicities of about seventy compounds containing N or O by means of the method CNDO/2. For the alkylamines, alcohols, ethers and carbonyl compounds, computational results agree qualitatively with the experimental values. The sequences of gas-phase basicities for the series of these compounds are as follows: Et2NH>Me3N>t-BuNH2>Me2NH>i-PrNH2>n-BuNH2>n-PrNH2>EtNH2>MeNH2>NH3; Et2O>EtOMe>t-BuOH>Me2O>i-PrOH>n-BuOH>n-PrOH>EtOH>MeOH>H2O; n-PrCHO>EtCHO>MeCHO>HCHO; n-BuCO2H>n-PrCO2H>EtCO2H>MeCO2H>HCO2H; HCO2Bu-n>HCO2Pr-N>HCO2Et>HCO2Me>HCO2H Obviously, alkyl substitution plays a role to increase the gas-phase basicities. The squence of increasing effectiveness is t-Bu>i-Pr>n-Bu>n-Pr>Et>Me For the amines containing heteroatoms investigated here, the gas-phase basicities have the following order repectively: CH3NH2>NH2NH2>NH2OH>NH2F>NHF2>NF3 The gas-phase basicities of these compounds change regularly with various substitutents. For the aliphatic compounds, the gas-phase basicity increases with thosizo and the degree of branching of the alkyl groups. For the amines containing heteroatoms, the gas-phase basicity decreases with increasing of the electro-negativity of the substitutent. For the relationship between the gas-phase basksity and the charge distribution and the ionization potentials, the conclusions are as follows: (1) The gas-phase basicities of the homologous compounds are proportional to the electron density of the atom N or O, but those of Rn NH3-n and Rn OH2-n are inversely proportional to the electron denisty of atom N or O. This shows that the base strength of the molecule cannot be determined solely by the electron density of the individual atom. (2) In the protonation reaction the alkyl groups spread the charges from the charged center. This effect enables protonated cations to become more stable because of the charge distribution av     

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.     

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.     

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.     

Prediction of the charge states of folded proteins in electrospray ionization

Earlier work from this laboratory dealt with the observation that the charge states of non-denatured proteins can be decreased by use of buffer salts in which the gas-phase basicity of conjugate base B, GB(B), of the buffer cations is high. A theoretical model was developed and applied to several small proteins. The predictions of the charge states were found to be in good agreement with those observed experimentally. Because the computational model is based on the charge residue model (CRM), the observed agreement lends support for the CRM. In the present work, the same model is applied to recent data by Catalina et al. who showed that very large charge reductions are achieved with very high GB(B) proton sponges. Their data included lysozyme but also the very much larger proteins, p-hydroxybenzoate hydroxylase (PHBH), 90 kDa and glutamate synthase (GLTS), 166 kDA. The present work examines the performance of the model for the much stronger bases and the very much larger proteins. It is found that the predictions of the charge states agree well for the small protein lysozyme but somewhat less well with the experimental results for PHBH and GLTS. The causes for the lack of good agreement with the large proteins are examined.     

Surface-induced dissociation of singly and multiply protonated polypropylenamine dendrimers

The ease of fragmentation of various charge states of protonated polypropylenamine (POPAM) dendrimers is investigated by surface-induced dissociation. Investigated are the protonated diaminobutane propylenamines [DAB(PA)n] DAB(PA)8 (1+ and 2+), DAB(PA)16 (2+ and 3+), and DAB(PA)32 (3+ and 4+). These ions have been proposed to fragment by charge-directed intramolecular nucleophilic substitution (SNi) reactions. Differences in relative fragment ion abundances between charge states can be related to the occupation of different protonation sites. These positions can be rationalized based on estimates of Coulomb energies and gas-phase basicities of the protonation/fragmentation sites. The laboratory collision energies at which the fragment ion current is approximately 50% of the total ion current were found to increase with the size, but to be independent of charge state of the protonated POPAM dendrimers. It is suggested that intramolecular Coulomb repulsion within the multiply protonated POPAM dendrimers selected for activation does not readily result in easier fragmentation, which is in accordance with the proposed fragmentation mechanism.     

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.     

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.     

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.     

Gas-phase protonation and deprotonation of acrylonitrile derivatives N[triple chemical bond]C--CH==CH--X (X=CH(3), NH(2), PH(2), SiH(3))

A combined experimental and theoretical study on the gas-phase basicity and acidity of a series of cyanovinyl derivatives is presented. The gas-phase basicities and acidities of (N[triple chemical bond]C--CH==CH--X, X=CH(3), NH(2)) were obtained by means of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry techniques. The corresponding calculated values were obtained at the G3B3 level of theory. The effects of exchanging CH(3) for SiH(3), and NH(2) for PH(2), were analyzed at the same level of theory. For the neutral molecules, the Z isomer is always the dominant species under standard gas-phase conditions at 298 K. The loss of the proton from the substituent X was found systematically to be much more favorable than deprotonation of the HC==CH linking group. The corresponding isomeric E ion is much more stable than the Z ion, so that only the former should be found in the gas phase. The most significant structural changes upon deprotonation occur for the methyl and amino derivatives because, in both cases, deprotonation of X leads to a significant charge delocalization in the corresponding anion. Protonation takes place systematically at the cyano group, whereby the isomeric E ion is again more stable than the Z ion. Push-pull effects explain the preference of aminoacrylonitrile to be protonated at the cyano group, which also explains the high basicity of this derivative relative to other members of the analyzed series that present rather similar gas-phase basicities, GB approximately 780 kJ mol(-1), indicating that the different nature of the substituents has only a weak effect on the intrinsic basicity of the cyano group. The cyanovinyl derivatives have a significantly stronger gas-phase acidity than that of the corresponding vinyl compounds CH(2)==CH--X. This acidity-strengthening effect of the cyano group is attributed to the greater stabilization of the anion with respect to the corresponding neutral compound.     

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.     

A model for solvated ion emission from electrospray droplets

A model is presented which shows that the energy required to emit small singly charged and large multiply charged (protein) solvated ions from electrospray droplets can be considerably lower than those predicted by earlier models. By allowing the droplet surface to distort in reaction to the emerging ion, a more nuanced picture of the ion emission mechanism appears, one that covers the range from pure ion evaporation (PIE) for small ions to what may be termed activated pseudo-Rayleigh ion release (PRIR), a mechanism that yields charge states nearly indistinguishable from the charge residue model (CRM), for large ions. Predictions based on this model are qualitatively consistent with many experimentally observed trends.     

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.     

Ion-Ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.     

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B(5) homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 degrees C and subunit concentrations ranging from 5 to 85 microM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 microM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B(5) ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 degrees C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B(5) ions at the +12 and +13 charge states were compared with values reported for the corresponding B(5) ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E(a)) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E(a) values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.     

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.