Gentle protein ionization assisted by high-velocity gas flow

Gentle protein electrospray ionization is achieved using the high-velocity gas flow of an air amplifier to improve desolvation in conventional ESI and generate intact folded protein ions in the gas phase. Comparisons are made between the ESI spectra of a number of model proteins, including ubiquitin, cytochrome c, lysozyme, and myoglobin, over a range of pH values under optimized conditions, with and without using an air amplifier to achieve high-velocity gas flow. Previously reported increased ion signals are confirmed. In addition, the peaks recorded using the air amplifier are shown to be narrower, corresponding to more complete desolvation. Significant changes in the charge-state distribution also are observed, with a shift to lower charge state at high-velocity flow. The relationship between the observed charge-state distribution and protein conformation was explored by comparing the charge-state shifts and the distributions of charge states for proteins that are or are not stable in their native conformations in low pH solutions. The data suggest retention of native or nativelike protein conformations using the air amplifier in all cases examined. This is explained by a mechanism in which the air amplifier rapidly creates small droplets from the original large ESI droplets and these microdroplets then desolvate without a significant decrease in pH, resulting in retention of the folded protein conformations. Furthermore, the holoform of ionized myoglobin is visible at pH 3.5, a much lower value than the minimum needed to see this form in conventional ESI. These results provide evidence for the importance of the conditions used in the desolvation process for the preservation of the protein conformation and suggest that the conditions achieved when using high-velocity gas flows to assist droplet evaporation and ion desolvation are much gentler than those in conventional ESI experiments.     

Gas-phase interference-free analysis of protein ion charge-state distributions: detection of small-scale conformational transitions accompanying pepsin inactivation

Analysis of protein ion charge-state distributions in electrospray ionization (ESI) mass spectra has become an indispensable tool in the studies of protein dynamics. However, applications of this technique have been thus far limited to detection of large-scale conformational transitions, which typically change the extent of multiple charging in a very significant way. However, more subtle conformational changes often elude detection, since the resulting changes of the extent of multiple charging are often smaller than the charge-state shifts caused by other external factors. Proton-transfer reactions involving protein ions and residual solvent molecules are the major extrinsic factors causing changes of charge-state distributions unrelated to conformational transitions. Since the extent of such reactions depends on the amount of various solvent components transferred to the ESI interface, profound changes of solvent composition may affect protein ion charge-state distributions not only by affecting protein higher order structure in solution but also through modulation of the efficiency of proton-transfer reactions in the gas phase. Here we demonstrate that it is possible to choose experimental conditions in such a way that the influence of gas-phase ion chemistry on protein ion charge-state distributions is not altered over a wide pH range. This methodology (gas-phase interference-free analysis of protein ion charge-state distributions, or GIFPICS) is sensitive enough to allow detection of pepsin inactivation under mildly acidic conditions. Pepsin is active and tightly folded in its native strongly acidic environment. Inactivation of pepsin at mildly acidic pH is not accompanied by global unfolding, as spectroscopic measurements suggest the protein remains compact. GIFPICS provides a means to observe this small-scale conformational transition that does not result in protein unfolding and may in fact elude detection by traditional spectroscopic techniques.     

Buffer loading for counteracting metal salt-induced signal suppression in electrospray ionization

The decrease in the sensitivity of electrospray ionization mass spectrometry caused by the presence of metal salts, such as sodium chloride, in the sample matrix is well known and is particularly problematic for biological samples. We report here that addition of high levels of ammonium acetate can improve analyte signal in aqueous electrospray solutions and counteracts the signal suppression caused by sodium chloride. A approximately 3-fold improvement in S/N is obtained by adding 8 M ammonium acetate to aqueous solutions of cytochrome c without added sodium chloride. No organic solvents or acids are added into the electrospray solutions. The signal-to-noise ratios of cytochrome c and ubiquitin (10(-)(5) M) ions formed from aqueous solutions containing 2.0 x 10(-)(2) M sodium chloride are improved by factors of approximately 7 and 11, respectively, by adding 7 M ammonium acetate to the solution. We propose that this effect is a result of the precipitation of Na(+) and Cl(-) from solution within the evaporating electrospray droplets prior to the formation of gas-phase protein ions. This method is potentially useful for improving the abundance of protein ions formed from solutions in which the molecules have a nativelike conformation and is particularly advantageous for such solutions that have high levels of sodium.     

In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.     

Effect of experimental parameters on the ESI FT-ICR mass spectrum of fulvic acid

Fulvic acid (FA) is a heterogeneous mixture of organic macromolecules found in the waters, soils, and sediments of the earth's surface. The ability of electrospray ionization (ESI) to effectively transfer large ions from the solution phase to the gas phase and the coupling of ESI to the high-mass-resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provide a potential method for the mass spectrometric analysis of FA. Positive- and negative-ion ESI FT-ICR MS analyses of four reference International Humic Substances Society FAs were performed. The spray solution composition was found to have a dramatic effect on the ion distributions, with high-mass aggregates (m/z approximately 2000-4000) being formed in less polar spray solutions. Positive-ion spectra for each FA obtained under optimum conditions resulted in number-average molecular weights ranging from 1700 to 1900. The mass spectra were extremely complex, with ion distributions on the order of m/z approximately 500-3000. The presence of more than one ion at each nominal mass was routinely observed. Negative-ion ESI analysis of the FA samples resulted in the observation of multiply charged ions whose distributions could be affected by the acidification of the spray solution. Solution parameters which have been reported to affect molecular weight distributions of FA such as pH, ionic strength, and concentration of multivalent cations were found to have little or no effect on the observed m/z distributions.     

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     

Ion trap collisional activation of the (M + 2H)2+ - (M + 17H)17+ ions of human hemoglobin beta-chain

The parent ions of human hemoglobin beta-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C-terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra.     

Estimates of protein surface areas in solution by electrospray ionization mass spectrometry

The extent of multiple charging of protein ions in electrospray ionization (ESI) mass spectra depends on the solvent-exposed surface area, but it may also be influenced by a variety of other extrinsic and intrinsic factors. Gas-phase ion chemistry (charge-transfer and charge-partitioning reactions) appears to be the major extrinsic factor influencing the extent of protonation as detected by ESI MS. In this work, we demonstrate that under carefully controlled conditions, which limit the occurrence of the charge-transfer reactions in the gas phase, charge-state distributions of protein ions can be used to assess the solvent-exposed surface area in solution. A set of proteins ranging from 5-kDa insulin to 500-kDa ferritin shows a clear correlation between the average charge in ESI mass spectra acquired under native conditions and their surface areas calculated based on the available crystal structures. An increase of the extent of charge-transfer reactions in the ESI interface results in a noticeable decrease of the average charge of protein ions across the entire range of tested proteins, while the charge-surface correlation is maintained. On the other hand, the intrinsic factors (e.g., a limited number of basic residues) do not appear to play a significant role in determining the protein ion charge. Based on these results, it is now possible to obtain estimates of the surface areas of proteins and protein complexes, for which crystal structures are not available. We also demonstrate how the ESI MS measurements can be used to characterize protein-protein interaction in solution by providing quantitative information on the subunit interfaces formed in protein associations.     

Electrospray ionization mass spectrometry of tetracycline, oxytetracycline, chlorotetracycline, minocycline, and methacycline

The effects of mobile-phase additives and analyte concentration on electrospray ionization mass spectra of a series of tetracyclines were investigated in both positive and negative ion modes. Only [M + H](+) and [M - H](-) ions were observed. The greatest sensitivity as [M + H](+) ions was obtained with 1% acetic acid and the greatest sensitivity as [M - H](-) ions was obtained using 50 mM ammonium hydroxide. Sensitivities in the positive ion mode were greater than those in the negative ion mode. The sensitivity as [M + H](+) showed no systematic variation with pH; however, the sensitivity as [M - H](-) did increase with increasing pH. A larger linear range was observed for [M - H](-) than for [M + H](+) ions. Both [M + Na](+) and [M + H](+) ions were observed with 0.5 mM sodium acetate and sodium iodide, but no adduct ions were observed with ammonium acetate. Some M(2)H(+) ions were observed at higher concentrations. Cluster ions, Na(NaOAc)(n)(+) or Na(NaI)(n)(+), but no sample ions were observed using 5 mM salts. The data suggest that mechanisms in addition to solution ionization are involved in the formation of the ESI sample ions. The utility of mobile phases containing 1% HOAc or 50 mM NH(4)OH was demonstrated for chromatographic separations.     

Electrosonic spray ionization. A gentle technique for generating folded proteins and protein complexes in the gas phase and for studying ion-molecule reactions at atmospheric pressure

Electrosonic spray ionization (ESSI), a variant on electrospray ionization (ESI), employs a traditional micro ESI source with supersonic nebulizing gas. The high linear velocity of the nebulizing gas provides efficient pneumatic spraying of the charged liquid sample. The variable electrostatic potential can be tuned to allow efficient and gentle ionization. This ionization method is successfully applied to aqueous solutions of various proteins at neutral pH, and its performance is compared to that of the nanospray and micro ESI techniques. Evidence for efficient desolvation during ESSI is provided by the fact that the peak widths for various multiply charged protein ions are an order of magnitude narrower than those for nanospray. Narrow charge-state distributions compared to other ESI techniques are observed also; for most of the proteins studied, more than 90% of the protein ions can be accumulated in one charge state using ESSI when optimizing conditions. The fact that the abundant charge state is normally as low or lower than that recorded by ESI or nanospray indicates that folded protein ions are generated. The sensitivity of the ionization technique to high salt concentrations is comparable to that of nanospray, but ESSI is considerably less sensitive to high concentrations of organic additives such as glycerol or 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base). Noncovalent complexes are observed in the case of myoglobin, protein kinase A/ATP complex, and other proteins. The extent of dissociation of protein ions in ESSI is comparable to or even smaller than that in the case of nanospray, emphasizing the gentle nature of the method. The unique features of ESSI are ascribed to very efficient spraying and the low internal energy supplied to the ions. Evidence is provided that the method is capable of generating fully desolvated protein ions at atmospheric pressure. This allows the technique to be used for the study of ion-molecule reactions at atmospheric pressure and examples of this are shown.     

Electrochemically induced pH changes resulting in protein unfolding in the ion source of an electrospray mass spectrometer.

The operation of an electrospray ion source in the positive ion mode involves charge-balancing oxidation reactions at the liquid/metal interface of the sprayer capillary. One of these reactions is the electrolytic oxidation of water. The protons generated in this process acidify the analyte solution within the electrospray capillary. This work explores the effects of this acidification on the electrospray ionization (ESI) mass spectrum of the protein cytochrome c (cyt c). In aqueous solution containing 40% propanol, cyt c unfolds around pH 5.6. Mass spectra recorded under these conditions, using a simple ESI series circuit, display a bimodal charge-state distribution that reflects an equilibrium mixture of folded and unfolded protein in solution. These spectra are not strongly affected by electrochemical acidification. An "external loop" is added to the ESI circuit when the metal needle of the sample injection syringe is connected to ground. The resulting circuit represents two coupled electrolytic cells that share the ESI capillary as a common anode. Under these conditions, the rate of charge-balancing oxidation reactions is dramatically increased because the ion source has to supply electrons for both, the external circuit and the ESI circuit. The analytical implications of this effect are briefly discussed. Mass spectra of cyt c recorded with the syringe needle grounded are shifted to higher charge states, indicating that electrochemical acidification has caused the protein to unfold in the ion source. The acidification can be suppressed by increasing the flow rate and lowering the electrolyte concentration of the solution and by using an electrolyte that acts as redox buffer. The observed acidification is similar for sprayer capillaries made of platinum and stainless steel. Removal of the protective oxide layer on the stainless steel surface results in effective redox buffering for a few minutes.     

Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.     

Solution Additives that Desalt Protein Ions in Native Mass Spectrometry

The presence of many salts, such as sodium chloride, can adversely affect the performance of native electrospray ionization mass spectrometry for the analysis of proteins and protein complexes by reducing the overall molecular ion abundances and distributing signal for any given charge state into many cationized forms with various numbers of adducts attached. Several solution additives, such as ammonium bromide, ammonium iodide, and NaSbF(6), can significantly lower the extent of sodium ion adduction to the molecular ions of proteins and protein complexes. For ubiquitin, addition of 25 mM ammonium bromide or ammonium iodide into aqueous solutions also containing 1.0 mM NaCl results in a factor of 72 and 56 increase, respectively, in the relative abundances of the fully protonated molecular ions compared to when these additives are not present. The effectiveness of this method for reducing sodium ion adduction is related to the low proton affinity (PA) values of the anions. Anions with very low PA also have a propensity to adduct as an acid molecule, but these adducts can be readily dissociated from the molecular ions either by activation in the source or subsequently by collisional activation in the mass spectrometer. This method of reducing sodium ion adduction to proteins is simple and requires no experimental modifications, making it an attractive alternative to other methods for desalting proteins prior to mass spectrometry analysis.     

Sonic spray as a dual polarity ion source for ion/ion reactions

A single sonic spray source has been used to generate both positive and negative ions for subsequent ion/ion reaction experiments. Ion/ion reactions took place after ions of each polarity were sequentially injected into a linear ion trap, where axial trapping was effected by applying an auxiliary radio frequency voltage to one end lens. Absolute charge reductions via proton transfer were demonstrated for multiply charged protein/peptide cations and multiply charged oligonucleotide anions. Deprotonation of polypeptide cations occurs with anions derived from fluorinated compounds such as nonadecafluoro-1-decanol and perfluoro-1-octanol, while multiply charged oligonucleotide anions are efficiently protonated via reaction with proton sponge (N,N,N',N'-tetramethyl-1,8-naphthalenediamine) cations. No evidence for signal suppression of the biopolymer ions was noted to result from the presence of these reagents in the solution subjected to sonic spray. Several of the analytically useful applications of ion/ion proton-transfer reactions are demonstrated using a single sonic spray ion source. These include an ion parking experiment for the purpose of gas-phase ion concentration and charge-state reduction of product ions formed via beam-type and in-trap collision-induced dissociation of multiply charged oligonucleotide parent anions. Examples of complex formation are also given to illustrate the flexibility of the sonic spray-induced ion/ion reaction method.     

Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification

A novel protocol for thermospray liquid chromatography/mass spectrometry (LC/MS) analysis of mixtures of glutathione conjugates is reported. Solvent conditions for optimal high-performance liquid chromatography are not always the same as for optimal thermospray ionization mass spectrometry. Labile glutathione conjugates that give poor spectra in aqueous ammonium acetate yield more intense molecular ion signals with increased percentages of acetonitrile. Direct injection thermospray ionization using 30-60% acetonitrile in aqueous ammonium acetate produced protonated molecular ions for glutathione conjugates of menadione, styrene oxide, pentachlorophenyl methyl sulfone, chlorodinitrobenzene, and chlorambucil. Since, the high percentages of organic modifier needed for good molecular ion intensity preclude chromatographic separation of these polar compounds, successful graphic separation of these polar compounds, successful LC/MS was facilitated by postcolumn addition of organic modifiers to the mobile phase. This new methodology allowed excellent chromatographic separations and thermospray ionization mass spectra to be obtained for a mixture of haloalkane glutathione conjugates. Moreover, cleavage of the gamma-glutamyl-cysteine amide bond of glutathione results in class-characteristic fragment ions. Changes in the fragmentation pathways in spectra acquired with and without organic modifiers shed light on the importance of the desolvation process in obtaining good molecular ion sensitivity in thermospray.     

Dual electrospray ion source for electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer

A dual electrospray ionization source (ESI) has been modified to simultaneously produce cations and anions, one from each emitter, for performing rapid electron-transfer dissociation (ETD) ion/ion reactions on a hybrid linear ion trap-orbitrap mass spectrometer. Unlike the pulsed dual ESI sources that were used to generate ETD reagent ions, this source separates the emitters in space, rather than time, by physically switching which one is in front of the atmospheric inlet. The new arrangement allows for substantially enhanced spray stability and decreased switching times (相似文献   

The Zeta Potential Op Droplets Prepared from a Self-Emulsifying Oil

The zeta potential of droplets formed by the self emulsification of compositions of n-hexane, phosphated nonylphenol ethoxylate and phosphated fatty alcohol ethoxylate has been determined in water and aqueous electrolyte solutions over a pH range of 3-11. Anomalies may be noted as a function of the n-hexane concentration of the surfactant/oil composition added to water. This, it is suggested, may be related to the mesomorphic systems that occur in bulk composition/water mixtures and could form at the interface of the droplets in water.

Effects on the electrophoretic/mobility observed in the presence of sodium, lithium, calcium or magnesium may be explained by the relative size of the sphere of hydration around the cat ion that controls the depth of penetration through the surface layers of the droplets.

Aluminum ions appear to react completely with the surfactant phosphate moiety, precipitating directly onto the droplet surface. The droplet surface charge always remains negative and below 30 mV except in the presence of aluminum ions. The maximum charge is obtained at the pH of 5.5 which approximates to the pKa₁ Df the surfactant mixture.     

Probing the unfolding and refolding processes of carbonic anhydrase 2 using electrospray ionization mass spectrometry combined with pH jump

A novel method for proving the time course of the unfolding and refolding processes of metalloprotein bovine carbonic anhydrase 2 (CA2) is demonstrated using electrospray ionization mass spectrometry (ESI MS) combined with pH jumps between 3.6 and 4.4. The shift in mass accompanied by the release or coordination of a zinc ion and the change in the charge state distribution were measured to evaluate the folding process. The time course of the ESI mass spectra revealed the existence of four types of ions in the experimental system, i.e., lower charged apo-CA2 and holo-CA2 ions and higher charged apo-CA2 and holo-CA2 ions. The deconvolution spectrum of the ion peak ensemble for each type of ion was processed and time course plots of the relative intensities of the four ions were prepared in order to analyze the folding processes. These analyses revealed the coexistence of two folding states of the lower and higher charged apo-CA2 under the condition of pH 3.6. The lower and higher charged apoproteins spontaneously refolded to the lower charged holoprotein by a pH jump from 3.6 to 4.4 without the addition of an extra zinc ion. The higher charged holoprotein observed during both the unfolding and refolding processes was considered to be an intermediate of the change in folding. The present study indicates that ESI MS combined with pH jump would be a powerful method to probe the unfolding and refolding of proteins. This method simultaneously measures mass spectra and analyzes the folding processes as a function of time using deconvolution spectra constructed by selecting a suitable m/z range for the analysis from the peaks of charge state distributions.     

Gd2O3:Eu phosphor powders prepared using a size-controllable droplet generator

Gd₂O₃:Eu phosphor powders were prepared by a filter expansion aerosol generator (FEAG) process capable of changing the mean size of droplets. The change in the mean size of the Gd₂O₃:Eu phosphor powders according to the concentrations of polyethylene glycol added to spray solutions was caused by the difference in the mean size of the droplets produced via the FEAG process. The mean sizes of droplets produced by the FEAG process were affected by the surface tension and viscosity of the spray solutions. The mean sizes of the Gd₂O₃:Eu phosphor powders obtained from the spray solutions with the same concentration of metal salts changed from 1.5 to 4.2 μm according to the concentrations of polyethylene glycol and citric acid added to the spray solutions. The maximum photoluminescent intensity of the phosphor powders obtained from the spray solutions with polymeric precursors and boric acid flux was 144% of that of the phosphor powders obtained from the aqueous spray solutions without flux.     

On the formation of highly charged gaseous ions from unfolded proteins by electrospray ionization

Electrospray ionization (ESI) of native proteins results in a narrow distribution of low protonation states. ESI for these folded species proceeds via the charged residue mechanism. In contrast, ESI of unfolded proteins yields a wide distribution of much higher charge states. The current work develops a model that can account for this effect. Recent molecular dynamics simulations revealed that ESI for unfolded polypeptide chains involves protein ejection from nanodroplets, representing a type of ion evaporation mechanism (IEM). We point out the analogies between this IEM, and the dissociation of gaseous protein complexes after collisional activation. The latter process commences with unraveling of a single subunit, in concert with Coulombically driven proton transfer. The subunit then separates from the residual complex as a highly charged ion. We propose that similar charge equilibration events accompany the IEM of unfolded proteins, thereby causing the formation of high ESI charge states. A bead chain model is used for examining how charge is partitioned as protein and droplet separate. It is shown that protein ejection from differently sized ESI droplets generates a range of protonation states. The predicted behavior agrees well with experimental data.