Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics

A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

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.     

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.     

Multiplexed MS/MS in a quadrupole ion trap mass spectrometer

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.     

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.     

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.     

Ionization and fragmentation of humic substances in electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was combined with ultra-high-resolution Fourier transform-ion cyclotron resonance mass spectrometry (FTICR MS) to characterize complex humic and fulvic acid mixtures. Lower than expected molecular weight distributions previously observed for humics when analyzed by ESI-MS have fueled speculation about a bias in favor of low molecular weight. Multiply charged ions, ionization suppression, and sample fragmentation have all been suggested as sources of this low molecular weight bias. In this work, resolution of the individual components of humic mixtures within a 1 mass-to-charge unit window was accomplished by FTICR MS at 9.4 T. At mass resolving powers between 60,000 (high mass) and 120,000 (low mass), it was possible to determine that virtually all ions present in spectra of Suwannee River fulvic and humic acid are singly charged, thus eliminating inadequate accounting for multiply charged ions as a primary source of any low molecular weight bias. The high-resolution mass spectra also revealed the presence of molecular families containing ions that differ from each other in degree of saturation, functional group substitution (primarily CH vs N and CH4 vs O), and number of CH2 groups. Ionization suppression and ion fragmentation were addressed for humic and fulvic acid mixtures and well-characterized poly(ethylene glycol) (PEG) mixtures with average molecular weights of 8000 and 10,000. Although these high molecular weight PEG mixtures fragment extensively under traditional positive-ion mode ESI conditions, similar fragmentation could not be confirmed for humic and fulvic acid mixtures.     

Atmospheric pressure photoionization fourier transform ion cyclotron resonance mass spectrometry for complex mixture analysis

We have coupled atmospheric pressure photoionization (APPI) to a home-built 9.4-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Analysis of naphtho[2,3-a]pyrene and crude oil mass spectra reveals that protonated molecules, deprotonated molecules, and radical molecular ions are formed simultaneously in the ion source, thereby complicating the spectra (>12 000 peaks per mass spectrum and up to 63 peaks of the same nominal mass), and eliminating the "nitrogen rule" for nominal mass determination of number of nitrogens. Nevertheless, the ultrahigh mass resolving power and mass accuracy of FT-ICR MS enable definitive elemental composition assignments, even for doublets as closely spaced as 1.1 mDa (SH3(13)C vs (12)C4). APPI efficiently ionizes nonpolar compounds that are unobservable by electrospray and allows nonpolar sulfur speciation of petrochemical mixtures.     

Multiplexed rectilinear ion trap mass spectrometer for high-throughput analysis

A multichannel mass spectrometer based on the rectilinear ion trap (RIT) analyzer was designed and constructed for simultaneous high-throughput analysis of multiple samples. The instrument features four parallel ion source/mass analyzer/detector channels assembled in a single vacuum chamber and operated using a common set of control electronics, including a single rf amplifier and transformer coil. This multiplexed RIT mass spectrometer employs an array of four millimeter-sized ion traps (x(o) = 5.0 mm and y(o) = 4.0 mm, where x(o) and y(o) are the half-distances in the x and y dimensions, respectively). Mass spectra are acquired from four different samples simultaneously. The available mass/charge range is m/z 15-510 with excellent linearity of the mass calibration (R2 = 0.999 999). The peak width is less than 0.3 mass/charge units at m/z 146, corresponding to a resolution of approximately 500. Simultaneous MS/MS of ions due to four compounds (3-fluoroanisole, 4-fluoroanisole, 2-fluorobenzyl alcohol, 2,6-dimethylcyclohexanone) with the same nominal molecular radical cation but distinctive fragmentation patterns was demonstrated. Isolation and fragmentation efficiencies were approximately 25 and approximately 75%, respectively, measured in the typical case of the molecular radical cation of acetophenone. Preacquisition differential data were obtained by real-time subtraction of the ion signals from two channels of the multiplexed mass spectrometer. The differential experiment presented offers proof of principle of comparative mass spectra in high-throughput screening applications while reducing data storage requirements.     

High-resolution field desorption/ionization fourier transform ion cyclotron resonance mass analysis of nonpolar molecules

We report the first field desorption ionization broadband high-resolution (m/Deltam(50%) approximately 65 000) mass spectra. We have interfaced a field ionization/field desorption source to a home-built 9.4-T FT-ICR mass spectrometer. The instrumental configuration employs convenient sample introduction (in-source liquid injection) and external ion accumulation. We demonstrate the utility of this configuration by generating high-resolution positive-ion mass spectra of C(60) and a midboiling crude oil distillate. The latter contains species not accessible by common soft-ionization methods, for example, low-voltage electron ionization, electrospray ionization, and matrix-assisted laser desorption/ionization. The present work demonstrates significant advantages of FI/FD FT-ICR MS for analysis of nonpolar molecules in complex mixtures.     

An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for "bottom-up" proteomics

Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows sub-second positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in a microLC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed.     

Ultrahigh Mass Accuracy in Isotope-Selective Collision-Induced Dissociation Using Correlated Sweep Excitation and Sustained Off-Resonance Irradiation: A Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Case Study on the [M + 2H](2+) Ion of Bradykinin

Using electrospray ionization with a 9.4 T Fourier transform mass spectrometer, fragment ion spectra were acquired for a single isotopomer of doubly protonated bradykinin (molecular mass, 1059.6 Da). Correlated sweep excitation methods were applied to mass-select the single isotopomer (m/z = 530.8). Sustained off-resonance irradiation was used to activate and fragment the ions. The accuracy (in terms of m/z) in detection of the fragment ions was on average 1.2 ppm, making the assignments unambiguous. The methods employed would be generally applicable to ions in the mass range of approximately 50 Da to 50 kDa.     

Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method

Direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (DI nESI FT-ICR MS) offers high mass accuracy and resolution for analyzing complex metabolite mixtures. High dynamic range across a wide mass range, however, can only be achieved at the expense of mass accuracy, since the large numbers of ions entering the ICR detector induce adverse space-charge effects. Here we report an optimized strategy for wide-scan DI nESI FT-ICR MS that increases dynamic range but maintains high mass accuracy. It comprises the collection of multiple adjacent selected ion monitoring (SIM) windows that are stitched together using novel algorithms. The final SIM-stitching method, derived from several optimization experiments, comprises 21 adjoining SIM windows each of width m/z 30 (from m/z 70 to 500; adjacent windows overlap by m/z 10) with an automated gain control (AGC) target of 1 x 10(5) charges. SIM-stitching and wide-scan range (WSR; Thermo Electron) were compared using a defined standard to assess mass accuracy and a liver extract to assess peak count and dynamic range. SIM-stitching decreased the maximum mass error by 1.3- and 4.3-fold, and increased the peak count by 5.3- and 1.8-fold, versus WSR (AGC targets of 1 x 10(5) and 5 x 10(5), respectively). SIM-stitching achieved an rms mass error of 0.18 ppm and detected over 3000 peaks in liver extract. This novel approach increases metabolome coverage, has very high mass accuracy, and at 5.5 min/sample is conducive for high-throughput metabolomics.     

Two-dimensional ECD FT-ICR mass spectrometry of peptides and glycopeptides

2D FT-ICR MS allows the correlation between precursor and fragment ions by modulating ion cyclotron radii for fragmentation modes with radius-dependent efficiency in the ICR cell without the need for prior ion isolation. This technique has been successfully applied to ion-molecule reactions, Collision-induced dissociation and infrared multiphoton dissociation. In this study, we used electron capture dissociation for 2D FT-ICR MS for the first time, and we recorded two-dimensional mass spectra of peptides and a mixture of glycopeptides that showed fragments that are characteristic of ECD for each of the precursor ions in the sample. We compare the sequence coverage obtained with 2D ECD FT-ICR MS with the sequence coverage obtained with ECD MS/MS and compare the sensitivities of both techniques. We demonstrate how 2D ECD FT-ICR MS can be implemented to identify peptides and glycopeptides for proteomics analysis.     

Modification of trapping potential by inverted sidekick electrode voltage during detection to extend time-domain signal duration for significantly enhanced fourier transform ion cyclotron resonance mass resolution

Applying an inverted voltage to the "sidekick" electrodes during ion cyclotron resonance detection improves both Fourier transform ion cyclotron resonance (FT-ICR) mass spectral signal-to-noise ratio (at fixed resolving power) and resolving power (at fixed signal-to-noise ratio). The time-domain signal duration increases by up to a factor of 2. The method has been applied to 7-T FT-ICR MS of electrosprayed positive ions from substance P and human growth hormone protein ( approximately 22 000 Da, m/Deltam50% 200 000), without the need for pulsed cooling gas inside the ICR trap. The modification can be easily adapted to any FT-ICR instrument equipped with sidekick electrodes. The present effects are shown to be comparable to electron field modification by injection of an electron beam during ICR detection, reported by Kaiser and Bruce (Kaiser, N. K.; Bruce, J. E. Anal. Chem. 2005, 77, 5973-5981.). Although the exact mechanism is not fully understood, computer simulations show that a flattening of the radial potential gradient along the magnetic field direction in the ICR trap may contribute to the effects. This study not only provides a way to enhance the quality of FT-ICR mass spectra but also offers insight into understanding of ion motions inside an ICR ion trap.     

Measuring the mass of an electron by LC/TOF-MS: a study of "twin ions"

While investigating the in-source CID fragmentation of nonsteroidal antiinflammatory drugs (NSAIDs), it was noticed that the same fragment ion (nominal mass) formed in either positive or negative ion electrospray for a suite of NSAIDs. For example, ibuprofen with a molecular mass of 206, fragments to the m/z 161 ion in negative ion from its deprotonated molecule (m/z 205, [M - H]-) and fragments to the m/z 161 ion in positive ion from its protonated molecule (m/z 207, [M + H]+). This fragment ion was euphemistically called a "twin ion"because of the same nominal mass despite opposite charge. The CID fragmentation of the twin ions was confirmed also by LC/MS/MS ion trap. Accurate mass measurements in negative ion show that the loss was due to CO2 (measured loss of 43.9897 atomic mass units (u) versus calculated loss of 43.9898 u for N = 10) and in positive ion the loss is due to HCOOH (measured loss of 46.0048 u versus calculated loss of 46.0055 u, N = 10). It was realized that, in fact, the ions were not "identical mass twins of opposite charge" but separated in accurate mass by two electrons. The accurate mass measurement by liquid chromatography/time-of-flight-mass spectrometry (LC/TOF-MS) can distinguish between the two fragment ions of ibuprofen (161.13362 +/- 0.00019 and 161.13243 +/- 0.00014 for N = 20). This experiment was repeated for two other NSAIDs, and the mass of an electron was measured as the difference between the twin ions, which was 0.00062 u +/- 14.8% relative standard deviation (N = 20 analyses). Thus, the use of continuous calibration makes it possible to measure the mass of an electron within one significant figure using the NSAID solution. This result shows the importance of including electron mass in accurate mass measurements and the value of a benchmark test for LC/TOF-MS mass accuracy.     

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     

Infrared multiple photon dissociation spectroscopy and computational studies of O-glycosylated peptides

The infrared multiple photon dissociation (IRMPD) spectra of O-glycosylated peptides in the gas phase were studied in the IR scanning range of 5.7-9.5 μm. Fragmentation of protonated and sodiated O-glycopeptides was investigated using electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with a free electron laser (FEL). FEL is used in the IRMPD technique as a tunable IR light source. In the IRMPD spectroscopic analysis of the protonated O-glycopeptide, fragment ions of the b/y and B/Y types were observed in the range of 5.7-9.5 μm, corresponding to the cleavage of the backbone in the parent amino acid sequence and glycosyl bonds, whereas the spectra of the sodiated glycopeptide showed major peaks of photoproducts of the B/Y type in the range of 8.4-9.5 μm. The IRMPD spectra of the O-glycopeptides were compared with simulated IR spectra for the structures obtained from the molecular dynamics.     

Electrothermal supercharging of proteins in native electrospray ionization

The formation of high charge-state protein ions with nanoelectrospray ionization (nESI) from purely aqueous ammonium bicarbonate solutions at neutral pH, where the proteins have native or native-like conformations prior to ESI droplet formation, is demonstrated. This "electrothermal" supercharging method depends on the temperature of the instrument entrance capillary, the nESI spray potential, and the solution ionic strength and buffer, although other factors almost certainly contribute. Mass spectra obtained with electrothermal supercharging appear similar to those obtained from denaturing solutions where charging beyond the total number of basic sites can be achieved. For example, a 17+ ion of bovine ubiquitin was formed by nESI of a 100 mM ammonium bicarbonate, pH 7.0, solution, which is three more charges than the total number of basic amino acids plus the N-terminus. Heating of the ESI droplets in the vacuum/atmosphere interface and the concomitant denaturation of the protein in the ESI droplets prior to ion formation appears to be the primary origin of the very high charge-state ions formed from these purely aqueous, buffered solutions. nESI mass spectra resembling those obtained under traditional native or denaturing conditions can be reversibly obtained simply by toggling the spray voltage between low and high values.