Profiling an electrospray plume using surface-enhanced Raman spectroscopy

We report the use of silver nanoparticles to obtain surface-enhanced Raman spectra of Crystal Violet in an electrospray plume. Surface enhancement allowed detection at low concentrations with the high specificity afforded by vibrational spectroscopy. SERS spectra were used to obtain an axial concentration profile closely matching that obtained in previous fluorescence experiments. SERS can provide more analyte structural information than has been obtainable from fluorescence studies of the plume.     

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.     

Conformational and noncovalent complexation changes in proteins during electrospray ionization

Electrospray ion sources efficiently produce gas-phase ions from proteins and their noncovalent complexes. Charge-state distributions of these ions are increasingly used to gauge their conformations in the solution phase. Here we investigate how this correlation is affected by the spraying conditions at the early stage of droplet generation, prior to the ionization process. We followed the folding behavior of model proteins cytochrome c and ubiquitin and the dissociation of the noncovalent holomyoglobin complex. Spray current measurements, fast Taylor cone imaging, and mass analysis of the generated ions indicated that the protein structure experienced conformational or complexation changes upon variations in the spraying mode of the electrospray ionization source. These effects resulted in a departure from the original secondary, tertiary, and quaternary structure of proteins, possibly introducing artifacts in related studies. Therefore, if a particular gas-phase ion conformation is required or correlations with the liquid-phase conformations are studied, it is advantageous to maintain a particular spraying mode. Alternatively, spraying mode-induced changes can be utilized to alter the structure of proteins in, for example, time-resolved experiments for the study of protein folding dynamics.     

The effect of small cations on the positive electrospray responses of proteins at low pH

Solutions consisting of protein and small molecule mixtures have been subjected to electrospray ionization to study the influence of small molecule/cation components at high concentrations on the electrospray responses of proteins. Emphasis was placed on solutions consisting of equal parts methanol and water and containing 1 vol % acetic acid. The results, therefore, are relevant to low pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. A variety of small cations/molecules were selected to sample a range of chemical characteristics. For example, sodium and cesium cations were studied to represent metal ions, tetrabutylammonium and tetramethylammonium cations were studied to represent quaternary ammonium compounds with different surface activities, and octadecylamine and glycine were studied to represent species that compete for protons but have different surface activities. A methodology for measuring relative ion suppression efficiencies was developed and applied for protein ions derived from bovine cytochrome c. The form of the small cation (i.e., metal ion, quaternary ammonium ion, or protonated molecule) did not appear to be a factor in determining the efficiency with which protein ion signals were suppressed. The extent to which ions are expected to concentrate on the surface, however, was the major factor in determining the ion suppression efficiency. Itwas found that the ion suppression efficiency of the most surface active species in this study was comparable to that of a protein on another protein after normalization by charge. These results are particularly relevant to the development of mixture analysis strategies based on ionization and tandem mass spectrometry applied to mixtures of whole proteins.     

Profiling of isovalertatin family aminooligosaccharides extracted from the culture of Streptomyces luteogriseus by using liquid chromatography/electrospray ionization mass spectrometry

A rapid and novel procedure using the liquid chromatography coupled to an electrospray ionization ion trap mass spectrometry technique was applied for the profiling of isovalertatin-family aminooligosaccharides in the extract from the culture filtrate of Streptomyces luteogriseus. The aminooligosaccharides were separated on a C-8 reversed-phase column with an acetonitrile-alkaline water gradient. The desired homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the characteristic fragment ions in their collision-induced dissociation spectra. This facile procedure led to the identification of all the five known aminooligosaccharides, isovalertatins M03, M13, M23, D03, and D23, in addition to the characterization of at least 41 novel isovalertatins, the molecular weights of which ranged from 729 to 2,793. This kind of assay should be intended as a simple and convenient way for the high-throughput analysis of screening aminooligosaccharides and potentially other structural families of natural products.     

gamma-Ray-mediated oxidative labeling for detecting protein conformational changes by electrospray mass spectrometry

Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of gamma-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as k(app) = k(RAD)/([P](tot) + C/k(u)), where k(RAD) is the primary rate of *OH formation, [P](tot) is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model by proposing that k(u) = [see text for formula] , where N is the number of amino acids, alphai is a measure for the solvent exposure of residue i, and k(ch)(i) is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H(3)PO(4) increases k(app) by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall *OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known to be minimal and where [P](tot) is constant.     

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.     

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 was performed by means of a rapid and facile procedure using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC/ESI-MS). The acarviostatins were separated on a C18 UPLC column with a series of acetonitrile-aqueous ammonia gradients. The target homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the diagnostic fragment ions in their MS/MS spectra. All six known reference acarviostatins (I03, II03, II13, II23, III03, IV03) were thus identified. In addition, at least 74 acarviostatin homologues, including 65 novel compounds, were characterized. Some of the features of the novel structures included having up to five acarviosine moieties, an acarviosine moiety at the reducing terminus, or an incomplete acarviosine moiety at the nonreducing terminus. This type of investigation may be useful for researchers who study secondary metabolomics in microorganisms and plants, especially those who perform metabolic profiling of aminooligosaccharides and other natural products with similar structures.     

Thyroid dosimetry in the western trace of the Chernobyl accident plume

According to World Health Organization guidelines (WHO/SDE/PHE/99.6), the reference level for consideration in stable iodine prophylaxis is based on the inhalation exposure pathway. In the western trace of the Chernobyl accident, the measurement of airborne (131)I fractions (aerosol-associated, gaseous reactive and gaseous organic) indicates that airborne gaseous reactive and, especially, organic (131)I fractions were the major contributors to thyroid exposure due to inhalation. The contribution of inhaled short-lived radioiodines was negligible. To attain more precise thyroid exposure evaluation, (131)I dose factors were determined as a function of age and prevalence of stable iodine deficiency. The results demonstrate that children with a stable iodine deficiency experienced at least two times higher thyroid doses than did children with a dietary iodine sufficiency. The results of these investigations demonstrate that in thyroid dosimetry it is important to know the stable iodine status as well as to have a standardised method for airborne radioiodine measurements, especially for consideration of stable iodine prophylaxis based on the inhalation exposure pathway.     

Probing the stability of biocompatible sodium hyaluronate/chitosan nanocoatings against changes in salinity and pH

The stability of polyelectrolyte multilayer assemblies was investigated with emphasis on the effects of solution ionic strength, pH, and polymer molecular weight on the film thickness and surface topography. The multilayers consisting of two polysaccharides, the polyanion sodium hyaluronate (HA) and the polycation chitosan (CH) were studied using surface plasmon resonance (SPR) spectroscopy, impedance quartz crystal microbalance (QCM), and atomic force microscopy (AFM). SPR/QCM experiments show that coatings consisting of four HA/CH bilayers assembled at pH 4.5 in the presence of 0.15 M NaCl are stable in NaCl solutions of concentration less than 0.8 M. These multilayers are stable when placed in contact with aqueous solutions ranging in pH from 3.5 to 9. The molecular weight of the polysaccharides has only a marginal effect on the stability of the films in the range explored here (HA: Mn = 360,000 or 31,000 g/mol; CH: Mn = 160,000 or 30,000 g/mol). AFM imaging reveals that different mechanisms may account for the multilayers stability versus salt and pH treatments. While increasing the ionic strength induces reorganization of the surface topography from isolated spherical islets to elongated worm-like features, changes in pH have no appreciable effects on the coating topography prior to complete disintegration.     

Mixed convection in wall plume of power-law fluids

Summary The non-similar boundary layer solutions are presented to study the mixed convective flow of a power-law fluid above a vertical adiabatic surface with a steady thermal source at the leading edge. The boundary layer equations contain an important mixed convection parameter. The governing non-similar equations are solved by means of a novel finite difference scheme for several values of the power-law viscosity index, the buoyancy parameter and the non-Newtonian Prandtl number of the fluid. The solutions obtained are uniformly valid over the entire regime of the buoyancy parameter ranging from forced convection to free convection limits. Consideration is given to the case of buoyancy-assisted and buoyancy-opposed plumes.     

Ambient gas influence on electrospray potential as revealed by potential mapping within the electrospray capillary

The effect of ambient gas on potentials inside the electrospray (ES) capillary was investigated. Potential measurements and differential electrospray emitter potential (DEEP) maps were obtained with the help of a small, movable, disklike platinum wire electrode inserted into the ES capillary. Typical solvents used for electrospray mass spectrometry such as methanol and mixtures of methanol-water and chloroform/methanol have been tested. It was found that oxygen is readily adsorbed from the surrounding ambient gas into the spraying liquid. Following adsorption, it resides in, or near to, the Taylor cone, thereby affecting the electrochemical potential near the ES capillary exit, as well as the character of the inherent electrochemical reactions occurring during the ES process. The potentials measured in an air environment with reactive oxygen present are contrasted against those obtained in an inert nitrogen environment. The kinetics of oxygen admission have been found to be quite fast, i.e., occurring in a matter of seconds, but it takes far longer to purge the system of oxygen by changing the ambient atmosphere to nitrogen. The oxygen effect is present in negative and positive ion modes of ES, but the total ES current is not affected by the change of ambient gas. The magnitude of the oxygen effect owing to ambient air was compared to the effect caused by initially dissolving oxygen in the solution prior to the start of ES; it was found that the presence of oxygen in the ambient gas has a far greater consequence. These results indicate that the presence of reactive gases, such as molecular oxygen, in the region of the ES emitter may have unintended secondary effects on the ES process prior to mass spectrometric analysis.     

Absorption and emission spectra of the YBCO laser plume

The plasma produced during laser ablation deposition of thin film YBCO has been studied by optical emission spectroscopy. There is evidence of increased YO band emission in the range 590–625 nm as the ambient oxygen gas pressure confining the plume is increased in the range 30–200 m Torr. Temporal profiles show that close to the target the plume is insensitive to ambient oxygen pressure. It is deduced that the optical emission here is excited by electron impact excitation. Further away from the target there is evidence that two distinct processes are at work. One is again electron excitation; the emission from this process decreases with distance because the expanding plume cools and collisions become less frequent in the expanding gas. The second is driven by oxidation of atomic species expelled at high speeds from the target. The main region of this activity is in the plume sheath where a shock front ensures heating of ambient O₂ and reaction of monatomic plasma species to form oxide in an exothermic reaction. Spatial mapping of the emission demonstrates clearly how increasing oxygen gas pressure confines the plasma and enhances the emission intensity from the molecular YO species ejected from the target in a smaller region close to the target. Ba⁺ is observed as a dominant species only very close to (within 1 mm of) the target. Absorption spectra have been taken in an attempt to examine ground state and cool species in the plume. They reveal the quite surprising result that YO persists in the chamber for periods up to 1 msec. This suggests an explanation for the recent report of off-axis laser deposition in terms of simple condensation. Previously, quasi-ballistic transfer of material from target to substrate has been considered the only significant process.     

Simultaneous detection of pH changes and histamine release from oxyntic glands in isolated stomach

Real-time simultaneous detection of changes in pH and levels of histamine over the oxyntic glands of guinea pig stomach have been investigated. An iridium oxide pH microelectrode was used in a potentiometric mode to record the pH decrease associated with acid secretion when the sensor approached the isolated tissue. A boron-doped diamond (BDD) microelectrode was used in an amperometric mode to detect histamine when the electrode was placed over the tissue. Both sensors provided stable and reproducible responses that were qualitatively consistent with the signaling mechanism for acid secretion at the stomach. Simultaneous measurements in the presence of pharmacological treatments produced significant variations in the signals obtained by both sensors. As the H2 receptor antagonist cimetidine was perfused to the tissue, histamine levels increased that produced an increase in the signal of the BDD electrode whereas the pH sensor recorded a decrease in acid secretion as expected. Addition of acetylcholine (ACh) stimulated additional acid secretion detected with the pH microelectrode whereas the BDD sensor recorded the histamine levels decreasing significantly. This result shows that the primary influence of ACh is directly on the parietal cell receptors rather then the ECL cell receptors of the oxyntic glands. These results highlight the power of this simultaneous detection technique in the monitoring and diagnosis of physiological significant signaling mechanisms and pathways.     

Characterization of local pH changes in brain using fast-scan cyclic voltammetry with carbon microelectrodes

Transient local pH changes in the brain are important markers of neural activity that can be used to follow metabolic processes that underlie the biological basis of behavior, learning and memory. There are few methods that can measure pH fluctuations with sufficient time resolution in freely moving animals. Previously, fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the measurement of such pH transients. However, the origin of the potential dependent current in the cyclic voltammograms for pH changes recorded in vivo was unclear. The current work explored the nature of these peaks and established the origin for some of them. A peak relating to the capacitive nature of the pH CV was identified. Adsorption of electrochemically inert species, such as aromatic amines and calcium could suppress this peak, and is the origin for inconsistencies regarding in vivo and in vitro data. Also, we identified an extra peak in the in vivo pH CV relating to the presence of 3,4-dihydroxyacetic acid (DOPAC) in the brain extracellular fluid. To evaluate the in vivo performance of the carbon-fiber sensor, carbon dioxide inhalation by an anesthetized rat was used to induce brain acidosis induced by hypercapnia. Hypercapnia is demonstrated to be a useful tool to induce robust in vivo pH changes, allowing confirmation of the pH signal observed with FSCV.