Michael Peschke

On the mechanisms by which the charged droplets produced by electrospray lead to gas phase ions

Abstract A brief description of the process leading to the formation of the very small droplets which ultimately produce gas phase ions is followed by a discussion of the ion evaporation model (IEM) and the charge residue model (CRM). The IEM developed for small ions by Iribarne and Thomson is a well developed model which provides quantitative predictions for the rates of evaporation of ions. The CRM attributed to Dole and extended by Rollgen does not provide a detailed consideration as to how the ‘final’ droplets containing only one ion are formed. Several experimental tests of IEM and CRM are discussed: The rates of formation of gas phase alkali ions M + (Li + , Na + , K + , Cs + ) determined from mass spectra, are compared with predicted rates by the Iribarne and Thomson equation. Unfortunately, the available thermochemical data required for the evaluation of the theoretical rates are of insufficient accuracy. Therefore, the theoretically predicted rates exhibit a large scatter. However, also, the experimentally determined relative rates on the basis of mass spectra are not in agreement. Relative rates for the alkali ions from this laboratory predict nearly equal rates for the alkali ions while data by Leize et al. predict an increase from Li + to Cs + . Both sets of data are compatible with the theoretical predictions of the IEM equation. The CRM, when extended to include the effect of surface activity of ions, is compatible with the results of nearly equal rates, but not compatible with the increasing rates of Leize et al . It is very desirable to establish in future work as to which set of experimental data is correct. If the increasing rates of Leize et al. are found to be true, a strong argument in favor of the IEM will be provided. Mass spectrometric observations of the intensities of the ions Na + and Na(NaCl) n + obtained from aqueous solutions of NaCl at different concentrations are compared with ion distributions expected on the basis of IEM and CRM. In general, larger intensities of Na + relative to Na(NaCl) n + would be expected on the basis of IEM. The experimental data are found to correspond much more closely to the predictions of IEM. However, various assumptions had to be made in order to be able to make the predictions. Most important of these is the history of the droplets as they undergo Rayleigh fission, and in particular, the size, charge and number of offspring droplets. Since accurate values for these quantities are not available, the conclusions in favor of IEM are not definitive. The final stages of CRM where droplets have radii of a few nanometers and several charges and solute molecules cannot be treated with the Rayleigh equation. Loss of single charges (ions) is possible in this stage. If this is the case, the last stage of CRM will be IEM-like. Such a stage will lead to a blurring of the distinction between IEM and CRM, for small ions. Fernandez de la Mora has provided very strong evidence that globular, not denatured, proteins are produced by CRM. More open, multiply-charged macroions could be produced by either IEM or CRM. The ions produced in the gas phase are not necessarily those present in the solution. Stable ions like the alkali ions are transferred without change to the gas phase. However, protonated bases may undergo changes as a consequence of the different basicity orders in solution and in the gas phase and other processes.

Hydration Energies and Entropies for Mg2+, Ca2+, Sr2+, and Ba2+ from Gas-Phase Ion−Water Molecule Equilibria Determinations

The sequential enthalpies , free energies , and entropies for the hydration reaction M(H2O)n-12+ + H2O = M(H2O)n2+ were determined in the gas phase for M = Mg, Ca, Sr, Ba. The gas-phase ion hydrates were produced by electrospray, and the hydration equilibria were determined in a reaction chamber attached to a mass spectrometer. The exothermicities of the (n − 1, n) reactions at low n (n = 1 to n = 5) are very high, and the corresponding equilibria require very high temperatures and could not be determined. For these low n, good theoretical results are available for Mg2+ and Ca2+ (Siegbahn and co-workers). A combination of the theoretical data with the experimental results (from n = 6 to n = 14) provides information on the inner and outer hydration shell structure and energetics of the hydrates. Very good agreement is observed between the theoretical and experimental energies where they overlap. For Mg, Ca, and Sr the first six molecules go into the inner shell while the seventh and higher molecules go int...

Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method

Several factors, attributable to the ESIMS mechanism, that can affect the assumptions of the titration method are examined: (1) The assumption that the concentrations in solution of the protein P, the ligand L, and the complex PL are proportional to the respective ion intensities observed with ESIMS, is examined with experiments in which ion intensities of two non-interacting proteins are compared with the respective concentrations. The intensities are found to be approximately proportional to the concentrations. The proportionality factors are found to increase as the mass of the protein is decreased. Very small proteins have much higher intensities. The results suggest that it is preferable to use only the intensity ratio of PL and P, whose masses are very close to each other when L is small, to determine the association constant KA in solution. (2) From the charge residue model (CRM) one expects that the solution will experience a very large increase of concentration due to evaporation of the precursor droplets, before the proteins P and PL are produced in the gas phase. This can shift the equilibrium in the droplets: P + L = PL, towards PL. Analysis of the droplet evaporation history shows that such a shift is not likely, because the time of droplet evolution is very short, only several μs, and the equilibrium relaxation time is much longer. (3) The droplet history shows that unreacted P and L can be often present together in the same droplet. On complete evaporation of such droplets L will land on P leading to PL and this effect will lead to values of KA that are too high. However, it is argued that mostly accidental, weakly bonded, complexes will form and these will dissociate in the clean up stages (heated transfer capillary and CAD region). Thus only very small errors are expected due to this cause. (4) Some PL complexes may have bonding that is too weak in the gas phase even though they have KA values in solution that predict high solution PL yields. In this case the PL complexes may decompose in the clean up stages and not be observed with sufficient intensity in the mass spectrum. This will lead to KA values that are too low. The effect is expected for complexes that involve significant hydrophobic interaction that leads to high stability of the complex in solution but low stability in the gas phase. The titration method is not suited for such systems.

Origin and number of charges observed on multiply-protonated native proteins produced by ESI

Abstract Native proteins and particularly native non-covalently bonded protein–protein and protein–substrate complexes are of great interest and are intensely studied by ESI–MS methods. The multiple charges on these ions are not only useful in lowering the m / z values but play also an important role in the chemical behavior of these complexes. Evidence from the literature and the present work is presented which supports the charge residue model (CRM) as the mode of formation of the charged globular proteins in the gas phase. Very small water droplets which contain only one protein molecule are ultimately formed in the ESI process. The surface of these droplets is charged by an excess of small ions due to a salt which is also present in the solution. Thus, in the positive ion mode, and when the buffer (ammonium acetate) is the main electrolyte used, the excess small positive ions are NH 4 + ions. Evaporation of the water in the droplet leads to a residue which is the globular protein. The protein is charged by the excess positive ions such as NH 4 + . The number of NH 4 + ions available, Z CRM , can be predicted on the basis of CRM. The proteins in order to be able to hold all of the protons provided must have a sufficient number of basic side chains located at the surface of the protein. It is found that most proteins have more than enough basic sites to hold the charge, Z CRM . Examples for these are carbonic anhydrase and cytochrome c . For these proteins the charge observed with ESI–MS is found to be close to equal to the charge, Z CRM , supplied. Some unusual proteins such as pepsin, have too few basic side chains, much less than the number of charges, Z CRM , provided. For these proteins the number of basic sites available on the protein determine how many of the charges provided by CRM will be retained. The number of basic sites can be evaluated and is found in agreement with the observed charges in the mass spectrum. Other predictions can also be made on the basis of the CRM. Thus, evaporation of the water droplet will lead to formation of neutral (uncharged) adducts on the protein, which are due to neutral components of the buffer. The approximate number of adducts can be predicted. Predictions can also be made which buffers will lead to adducts difficult to get rid off, in the desolvation stage of the mass spectrometer.

Formation, acidity and charge reduction of the hydrates of doubly charged ions M2+ (Be2+, Mg2+, Ca2+, Zn2+)

Abstract There are two methods for producing in the gas phase doubly charged metal ion hydrates, M(H 2 O) n 2+ (or other ion ligand L ML n 2+ complexes). In the clustering method, one starts with the naked ion M 2+ , and in the presence of a third (bath) gas and water vapor, the ion hydrates form by ion-molecule clustering reactions. The second method is based on electrospray with which a spray of aqueous solutions containing the dissolved salts M 2+ + 2X − , leads to gas phase M(H 2 O) n 2+ with a distribution around n ≈ 8. For M, which has a high second ionization energy, IE(M 2+ ), both methods can fail to produce a full range of hydrates with a given n , because of the interference of a charge reduction reaction which involves intramolecular proton transfer. This reaction becomes possible at n = 2; (M(H 2 O) 2 2+ )∗ = MOH + + H 3 O + , and competes with the simple ligand loss: (M(H 2 O) 2 2+ )∗ = M(H 2 O) 2+ + H 2 O. The thermally excited (M(H 2 O) 2 2+ )∗ results in the clustering method by the exothermicity of the forward clustering reaction and in the electrospray method by the thermal declustering required to produce lower n ions. Ab initio calculations are presented for the energies of the above reactions and transition states for Mg 2+ and Ca 2+ . These show that the transition state for the charge reduction reaction is much lower than that for the simple ligand loss at n = 2. However, as n increases, the two transition states move closer together and above a given n = r , simple ligand loss becomes dominant. The capabilities and limitations of the two methods to produce hydrates of a given n is discussed. Experimental results illustrate competing charge reduction and simple H 2 O loss for Be(H 2 O) n 2+ under thermal equilibrium conditions at n ≈ 9. Charge reduction reactions when occurring in the forward clustering direction can be viewed as proton transfer reactions to the incoming H 2 O molecule. These can be generalized by examining the proton affinities of the MOH(H 2 O) n + ions, which are obtained by ab initio calculations. Proton transfer from M(OH) 2 ) n 2+ can be induced not only by H 2 O but also by other bases B. Experimental results for the deprotonation of Zn(OH 2 ) n 2+ , n = 8 or 9, by NH 3 are presented. The charge reduction reactions by which a deprotonated ligand attached to M is formed, can have synthetic utility. Examples are given for the production of methylthiolate complexes which may be useful for modeling ion complexes in which one of the ligands is the deprotonated amino acid residue cysteine.

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.