Testing the role of solvent surface tension in protein ionization by electrospray

This work was aimed at probing the influence of solvent surface tension on protein ionization by electrospray. In particular, we were interested in testing the previously suggested hypothesis that the charge-state distributions (CSDs) of proteins in electrospray ionization mass spectrometry (ESI-MS) are controlled by the surface tension of the least volatile solvent component. In the attempt to minimize uncontrolled conformational effects, we used acid-sensitive proteins (cytochrome c and myoglobin) at low pH or highly stable proteins (ubiquitin and lysozyme) in the presence of low concentrations of organic solvents. A first set of experiments compared the effect of 1- and 2-propanol. These two alcohols have similar chemico-physical properties but values of vapor pressure below and above that of water, respectively. Both compounds have much lower surface tension than water. The solvents employed allowed testing of the influence of surface tension on protein spectra obtained from similarly denaturing solutions. The compared solvent conditions gave rise to very similar spectra for each tested protein. We then investigated the effect of the addition of dimethyl sulfoxide to acid-unfolded proteins. We observed enhanced ionization in the presence of acetic or formic acid, consistent with the previously described supercharging effect, but almost no shift of the CSD in the presence of HCl. Finally, we analyzed thermally denatured cytochrome c, to obtain reference spectra of the unfolded protein in high-surface-tension solutions. Also in this case, the CSD of the unfolded protein was shifted towards lower m/z values relative to low-surface-tension systems. In contrast to the other results reported here, this effect is consistent with an influence of solvent surface tension on CSD. The magnitude of the effect, however, is much smaller than predicted by the Rayleigh equation. The results presented here are not easy to reconcile with the hypothesis that the maximum charge state exhibited by proteins in ESI-MS reflects the Rayleigh-limit charge of the precursor droplet. The data are discussed with reference to models for the mechanism of electrospray ionization.     

Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry,circular dichroism and fluorescence spectroscopy

Electrospray ionization mass spectrometry (ESI‐MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI‐MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid‐ and organic solvent‐induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme–protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2–3.6, or solution containing 20–30% acetonitrile or 40–50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI‐MS conditions were established to observe the H₂O‐ and CO‐bound Mb complexes, respectively. H₂O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI‐MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI‐MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand‐binding complexes and characterizing protein conformational changes. Copyright © 2010 John Wiley & Sons, Ltd.     

Elucidating Factors Manipulated the Formation of Multiply Charged Protein Homomultimeric Complexes by Electrospray Ionization

Native non‐covalently bonded protein‐protein and protein‐substrate complexes are of great interest and have been extensively studied by electrospray ionization mass spectrometry (ESI‐MS). Multiply charged protein homomultimeric complexes are shown to form by ESI‐MS. This study addresses factors that can artificially induce the formation of multiply charged protein homomultimeric complexes. Cytochrome c (Cyt c) and ubiquitin, which are monomers in solution, were found to generate (Cyt c)_mⁿ⁺ by electrospray ionization (ESI). The homomultimeric complexes were not limited to dimeric complexes but include also multiply charged trimers, tetramers, and pentamers. The observation of these homomultimeric complexes has never been revealed from a Cyt c solution at the concentration as low as 10 μM. Increasing the concentration of Cyt c enhanced the formation of (Cyt c)_mⁿ⁺ as expected; however, the protein concentration does not affect the relative intensities of monomeric and dimeric complexes. Additionally the enrichment of NH₄OH also promotes the formation of (Cyt c)_mⁿ⁺. Notably, source collision‐induced dissociations (source‐CID) of (Cyt c)_mⁿ⁺ alter the charge state distribution (CSD) and may lead to an incorrect interpretation of Cyt c conformations. Hence, extra care should be taken when using CSD to interpret the conformation of a protein derived from ESI‐MS.     

Protein charge-state distributions in electrospray-ionization mass spectrometry do not appear to be limited by the surface tension of the solvent

According to a current model for protein electrospray, the charge-state distributions (CSDs) observed by electrospray-ionization mass spectrometry (ESI-MS) are controlled by the Rayleigh-limit charge of the droplets that generate the gas-phase protein ions. A testable prediction of this model is that the maximum charge state displayed by proteins in ESI-MS should respond to changes in the surface tension of the ESI droplets according to the Rayleigh equation. In this work, we subject this specific hypothesis to direct experimental testing. We show data obtained by time-of-flight (TOF) nano-ESI-MS with several different proteins in aqueous solutions containing 20-50% 1-propanol or 40% 1,2-propylene glycol. Both of these compounds have lower vapor pressure and lower surface tension than water. Propylene glycol also has a lower evaporation rate than water, providing an even more stringent test for surface tension effects in late ESI droplets. None of these cosolvents affects the CSDs of either folded or unfolded proteins as predicted by the Rayleigh-charge model. The only changes induced by 1-propanol can be ascribed to protein unfolding triggered above critical concentrations of the alcohol. Below such a threshold, no shift of the CSDs toward lower charge states is observed. The presence of 40% propylene glycol in the original protein solutions gives rise to CSDs that either are the same as those in the control samples or present much smaller changes than those calculated by the Rayleigh equation. Thus, the charge states of gas-phase protein ions produced by electrospray do not seem to be limited by the surface tension of the solvent. They rather appear to be quite protein-specific.     

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.     

Effects of pH on the kinetic reaction

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.     

Induction of protein conformational change inside the charged electrospray droplet

The behavior of the analyte molecules inside the neutral core of the charged electrospray (ES) droplet is not unambiguously known to date. The possibility of protein conformational change inside the charged ES droplet has been investigated. The ES droplets encapsulating the protein molecules were exposed to the acetic acid vapor in the ionization chamber to absorb the acetic acid vapor. Because of the faster evaporation of water than that of acetic acid, the droplets became enriched with acetic acid and thus altered the solvent environment (e.g. pH and polarity) of the final charged droplets from where the naked charged analytes (proteins) are formed. Thus, the perturbation of the ES droplet solvent environment resulted in the protein conformational change (unfolding) during the short lifespan of the ES droplet and that is reflected by the multimodal charge state distribution in the corresponding mass spectra. Further, the extent of this conformational change inside the ES droplet was found to be related to the structural flexibility of the protein. Although the protein conformational change inside the ES droplet has been driven by using acetic acid vapor in the present study, the results would help in the near future to understand the spontaneity of the conformational change of the analyte on the millisecond timescale of phase transition in the natural way of ES process. Copyright © 2013 John Wiley & Sons, Ltd.     

DILUTE SOLUTION BEHAVIOR OF CHITOSAN IN DIFFERENT ACID SOLVENTS

Dilute solution behavior of chitosan was studied in formic acid, acetic acid, lactic acid andhydrochloric acid aqueous solution under different pH values. The reduced viscosities, η_(sp)/C,ofchitosan solutions were dependent on the properties of acid and pH value of solvents. For a givenchitosan concentration, η~(sp)/C decreased with the increase of acid concentration, or decreasing pHof solvent, indicating shielding effect of excessive acid similar to adding salt into solution. Thestabilities of dilute chitosan solution in formic acid and lactic acid were better than that in acetic acid and hvdrochloric acid.     

Thermodynamic analysis of denatured lysozyme folded on moderately hydrophobic surface at 298 K

Both calorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of lysozyme (Lyz) denatured by 1.8 mol L⁻¹ guanidine hydrochloride (GuHCl) on a moderately hydrophobic packings at 298 K, pH 7.0 and various salt concentrations were carried out. Based on the thermodynamics of stoichiometric displacement theory (SDT) the fractions of thermodynamic functions, which related to four subprocesses of denatured protein refolding on the surface, were calculated and thermodynamic analysis that which one of the subprocesses plays major role for contribution to the thermodynamic fractions was made in detail. The moderately hydrophobic surface can provide denatured Lyz energy and make it gain more conformation with surface coverage or salt concentration increment. The displacement adsorptions of denatured Lyz onto PEG-600 surface are exothermic, more structure-ordered and enthalpy driven processes.     

Studies of the association of chitosan and alkylated chitosan with oppositely charged sodium dodecyl sulfate

Cationic biopolymer chitosan has many applications in food, cosmetic and pharmaceutical industries. Grafting alkylated chains on its backbone can hydrophobically modify this water-soluble polymer.This paper concerns unmodified chitosan, alkylated chitosan and their interactions with a model anionic surfactant, sodium dodecyl sulfate (SDS). The solvent is pH 4 acetic acid solution. The purpose of this study is to highlight the hydrophobicity brought by the alkylated chains by comparing surface tension measurements and rheological properties of hydrophobically modified polymer (HMP) and chitosan solutions at 25 °C.Interactions of chitosan and HMP with surfactant have also been investigated giving information about surface activity and electrical conductivity of such systems. It results that alkylated chitosan/SDS system is more surface active than chitosan/SDS and it offers new potential applications in pharmaceutical and cosmetic fields because of the formation of amphiphilic complexes.     

Is droplet evaporation crucial in the mechanism of electrospray mass spectrometry?

Evaporation of solvent from charged droplets was found not to be a prerequisite to ion desorption in electrospray mass spectrometry. Evidence of evaporation was absent in an examination of the electrospray mass spectral profiles of cytochrome c and myoglobin in 0.2% acetic and propionic acid solutions; the pHs of these two acid solutions are expected to change in opposite directions with evaporation. The results strongly suggest that ions, as observed in electrospray mass spectrometry, are desorbed from solutions that have undergone minimal evaporation, in other words, at the beginning rather than later parts of the electrospray process. It is speculated that ions are desorbed directly from the solution-air interface at the needle tip.     

The role of conformational flexibility on protein supercharging in native electrospray ionization

Effects of covalent intramolecular bonds, either native disulfide bridges or chemical crosslinks, on ESI supercharging of proteins from aqueous solutions were investigated. Chemically modifying cytochrome c with up to seven crosslinks or ubiquitin with up to two crosslinks did not affect the average or maximum charge states of these proteins in the absence of m-nitrobenzyl alcohol (m-NBA), but the extent of supercharging induced by m-NBA increased with decreasing numbers of crosslinks. For the model random coil polypeptide reduced/alkylated RNase A, a decrease in charging with increasing m-NBA concentration attributable to reduced surface tension of the ESI droplet was observed, whereas native RNase A electrosprayed from these same solutions exhibited enhanced charging. The inverse relationship between the extent of supercharging and the number of intramolecular crosslinks for folded proteins, as well as the absence of supercharging for proteins that are random coils in aqueous solution, indicate that conformational restrictions induced by the crosslinks reduce the extent of supercharging. These results provide additional evidence that protein and protein complex supercharging from aqueous solution is primarily due to partial or significant unfolding that occurs as a result of chemical and/or thermal denaturation induced by the supercharging reagent late in the ESI droplet lifetime.     

An efficient slurry packing procedure for the preparation of columns applicable in capillary electrochromatography and capillary electrochromatography‐electrospray‐mass spectrometry

A procedure is described for the slurry packing of 50‐μm ID fused silica capillaries with 3‐μm octadecyl silica (ODS) particles for capillary electrochromatography (CEC) and its hyphenation with electrospray ionisation mass spectrometry (ESI/MS). A homogeneous packed bed is obtained by using a slow packing process in an upward direction with a balanced density slurry solvent and MeOH as packing solvent. Special attention was paid to the in‐ and outlet frit preparation in order to avoid gas bubble formation which renders CEC‐ESI/MS problematic. Frits were made out of the packed bed itself, sintered in water, by using a perforated heating ribbon; they were not longer than 1 mm. In CEC‐UV, column efficiencies up to 300,000 plates per meter were obtained. Absence of gas bubbles was ascertained by the straightforward coupling to ESI/MS. A make‐up flow of 3 μL/min H₂O/MeOH containing 0.1% HCOOH was used in the sheath flow interface. Steroids and carbamates were analysed with a 0.1% triethylamine‐acetic acid buffer (pH 8.9) containing varying amounts of acetonitrile. In CE‐ESI/MS, efficiencies dropped by ca. 20% but spectral data were excellent.     

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to −80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution.     

Addressing a Common Misconception: Ammonium Acetate as Neutral pH “Buffer” for Native Electrospray Mass Spectrometry

Native ESI-MS involves the transfer of intact proteins and biomolecular complexes from solution into the gas phase. One potential pitfall is the occurrence of pH-induced changes that can affect the analyte while it is still surrounded by solvent. Most native ESI-MS studies employ neutral aqueous ammonium acetate solutions. It is a widely perpetuated misconception that ammonium acetate buffers the analyte solution at neutral pH. By definition, a buffer consists of a weak acid and its conjugate weak base. The buffering range covers the weak acid pK_a ± 1 pH unit. NH₄ ⁺ and CH₃-COO^? are not a conjugate acid/base pair, which means that they do not constitute a buffer at pH 7. Dissolution of ammonium acetate salt in water results in pH 7, but this pH is highly labile. Ammonium acetate does provide buffering around pH 4.75 (the pK_a of acetic acid) and around pH 9.25 (the pK_a of ammonium). This implies that neutral ammonium acetate solutions electrosprayed in positive ion mode will likely undergo acidification down to pH 4.75 ± 1 in the ESI plume. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. It is a volatile electrolyte that can mimic the solvation properties experienced by proteins under physiological conditions. Also, a drop from pH 7 to around pH 4.75 is less dramatic than the acidification that would take place in pure water. It is hoped that the habit of referring to pH 7 solutions as ammonium acetate “buffer” will disappear from the literature. Ammonium acetate “solution” should be used instead.

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Orientation of photosynthetic reaction center in Langmuir–Blodgett film by formation of cross-linked complex with cytochrome c

Cross-linked complex between photosynthetic reaction center and horse heart cytochrome c (cyt c) was prepared for the control of molecular orientation in Langmuir–Blodgett (LB) method. The surface of cyt c is highly hydrophilic whereas that of RC is hydrophobic. A polar distribution of hydrophobicity/hydrophilicity in the complex was realized by the cross-linkage of the different protein molecules. An index was introduced to evaluate the hydrophobicity of the surface of the proteins. The orientation of RCs in an LB film was evaluated by the displacement current. The complex showed a response 1.5 times larger than that of RC.     

Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was studied through their intrinsic fluorescence emission spectra, fluorescence phase diagrams, fluorescence quenching, size‐exclusion chromatographies, native polyacrylamide gel electrophoreses and deactivation profiles. The results showed that during their unfolding in urea and guanidine hydrochloride solutions, bovine heart cytochrome c molecules existed only in a unimolecular form and their bi‐molecular and/or poly‐molecular aggregates and aggregate precipitates were not formed all along. When the urea and guanidine hydrochloride concentrations in denaturation solution were separately about 6.0 and 3.0 mol/L, they could be completely deactivated and almost all of the tryptophan residues originally embedded in the interior of their molecules were exposed to the surface of their molecules. Different from the unfolding of the most often used horse heart cytochrome c, that of bovine heart cytochrome c induced by urea and guanidine hydrochloride was separately a completely co‐operative procedure and followed a two‐state model.     

The liquid–gas interface of oxygen–nitrogen solutions: 1. Surface tension

The capillary method of surface tension measurement has been used to measure the surface tension of oxygen–nitrogen solutions in the temperature range from 80 to 132 K. At temperatures below the nitrogen critical temperature (T_c = 126.2 K) the capillary constant and the surface tension of solutions are smaller than their additive values and vary linearly with the temperature. Experimental data are compared with the results of calculating the surface tension by the theories of Pinnes and Rowlinson.     

Thermodynamic Analysis of the Adsorption and Micellization Activity of the Mixtures of Rhamnolipid and Surfactin with Triton X-165

The surface tension of aqueous solutions of Triton X-165 with rhamnolipid or surfactin mixtures was measured. The obtained results were applied for the determination of the concentration and composition of the Triton X-165 and biosurfactants mixture at the water–air interface as well as the contribution of the particular component of the mixtures to water surface tension reduction and the mutual influence of these components on the critical micelle concentration. The determination of these quantities was based on both the commonly used concepts and a new one proposed by us, which assumes that the composition of the mixed monolayer at the water–air interface depends directly on the pressure of the monolayer of the single mixture component and allows us to determine the surface concentration of each mixture component independently of surface tension isotherms shape. Taking into account the composition of the mixed monolayer at the water–air interface, the standard Gibbs adsorption free energy was considered. The obtained results allow us to state that the concentration of both mixture components corresponding to their saturated monolayer and the surface tension of their aqueous solution can be predicted using the surfactants’ single monolayer pressure and their mole fraction in the mixed monolayer determined in the proposed way.