Change In Charge State Distribution Research Articles

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.

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.

Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS.

Monitoring the changes in charge-state distributions of protein ions in electrospray ionization (ESI) mass spectra has become one of the commonly accepted tools to detect large-scale conformational changes of proteins in solution. However, these experiments produce only qualitative, low-resolution information. Our goal is to develop a procedure that would produce quantitative data on protein conformational isomers coexisting in solution at equilibrium. To that end, we have examined the evolution of positive ion charge-state distributions in the

The methanol-induced conformational transitions of β-lactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry

The methanol-induced conformational transitions under acidic conditions for β-lactoglobulin, cytochrome c, and ubiquitin, representing three different classes of proteins with β-sheets, α-helices, and both α-helices and β-sheets, respectively, are studied under equilibrium conditions by electrospray ionization mass spectrometry (ESI-MS). The folding states of proteins in solution are monitored by the charge state distributions that they produce during ESI and by hydrogen/deuterium (H/D) exchange followed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induced α-helix structure. Intermediate states formed at about 35% methanol concentration are found to give bimodal charge state distributions. The same rate of H/D exchange is shown by the two contributions to the bimodal distributions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchange of proteins followed by ESI-MS shows that helical denatured states, populated at around 50% methanol concentration, transform into more protected structures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins.

Evidence of noncovalent dimerization of calmodulin.

Calcium-binding proteins, such as S-100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol. 2, 711-714; Kilby, P.M., Van Eldik, L.J. & Roberts, G. C. (1996) Structure 4, 1041-1052]. We have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium-binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra-high-resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5-10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its alpha-helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer-monomer equilibrium. The ultra-high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.

Radiation losses in PLT during neutral-beam and ICRF heating experiments

Radiation and charge-exchange losses in the PLT tokamak are compared for discharges with Ohmic heating only (OH), and with additional heating by neutral beams (NB) or RF in the ion cyclotron frequency range (ICRF). Spectroscopic, bolometric and soft-X-ray diagnostics were used. The effects of discharge cleaning, vacuum wall gettering, and rate of gas inlet on radiation losses from OH plasmas and the correlation between radiation from plasma core and edge temperatures are discussed. – For discharges with neutral-beam injection the radiation dependence on type of injection (e.g. co-injection versus counter- and co- plus counter-injection) was investigated. Radial profiles of radiation loss were compared with profiles of power deposition. Although total radiation was in the range of 30–60% of total input power into relatively clean plasma, nevertheless only 10–20% of the total central input power to ions and electrons was radiated from the plasma core. The radiated power was increased mainly by increased influx of impurities, however, a fraction of this radiation was due to the change in charge-state distribution associated with charge-exchange recombination. – During ICRF heating radiation losses were higher than or comparable to those experienced during co- plus counter-injection at similar power levels. At these low power levels of ICRF heating the total radiated power was ∼ 80% of auxiliary-heating power. Radiation losses changed somewhat less rapidly than linearly with ICRF power input up to the maximum available at the time of these measurements (0.65 MW).