Formation of Alternating Surfactant-Enriched and Surfactant-Depleted Phases in the Taylor Cone of a Nanoelectrospray.

The electrospray ionization of highly conductive solutions containing Triton X-100, a nonionic surfactant, is found to induce alternating periods of surfactant enrichment and depletion when the concentration of the surfactant is near the critical micelle concentration (CMC) and when the flow rate is on the order of 10 nL/min. Analyzing the surfactant-protein mixture shows that the protein is partially denatured during the surfactant enrichment. The measurement of the phospholipid and oligosaccharide mixture prepared in the surfactant solution shows that the ion signal of the lipid is in phase with, and the hydrophilic oligosaccharide is out of phase with the surfactant signal. The results suggest that this novel phenomenon can be exploited for in situ separation of compounds in ESI-MS. Besides the ion signal, the condition of the alternating phase is also reflected in the spray current and Taylor cone's apex angle. The phase separation is likely related to the formation of a micelle in the Taylor cone and can be selectively triggered by tuning the flow rate with emitter voltage for an on-demand application.

Tuning the Internal Energy of Ions Produced by Atmospheric and High-Pressure Electrospray by Modulating the Gas Throughput into the First Vacuum Stage.

In a high-pressure environment, electrospray ionization (ESI) can be achieved without discharge between the emitter and the counter electrode, thus enabling the generation of gas-phase ions from liquid with high surface tension, such as pure water, which requires a high onset voltage for stable ESI. In this study, the ion dissociation during the transferring of ions/charged droplets from a superatmospheric pressure environment to vacuum has been systematically investigated using benzyl ammonium thermometer ions. The ion source pressure did not affect the internal energy distribution of ions, whereas the gas throughput into the first vacuum stage clearly influences the internal energy distribution of the ions. The increase in the gas throughput increased the density of molecules/atoms presented in ion transfer/focusing electrodes located in the first vacuum stage. As a result, the mean free path of ions in the first vacuum stage decreases, and the energy of ions decreases by decreasing the kinetic energy involved in each collision between ions and residue gas. The gas throughput into the first vacuum stage is found to describe the internal energy distribution of ions associated with the local conditions more quantitatively instead of using the measured pressure of the vacuum stage, which is different from the effective local pressure. This study also demonstrated the controlled dissociation of ions using the ion transfer settings of the instrument in combination with ion inlet tubes of different sizes.

Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics.

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

Probing Acid-Induced Compaction of Denatured Proteins by High-Pressure Electrospray Mass Spectrometry.

Further increase in the acidity in the most denaturing acidic solution is known to induce compaction of the fully unfolded protein into a compact molten globule. The phenomenon of "acid-induced folding of proteins" takes place at pH ∼1 in strong acid aqueous solutions with high electrical conductivity and surface tension, a condition that is difficult to handle using conventional electrospray ionization methods for mass spectrometry. Here, high-pressure electrospray ionization (HP-ESI) is used to produce well-resolved mass spectra for proteins in strong acids with pH as low as 1. The compaction of protein conformation is indicated by a large shift in the charge state from high charges to native-like low charges. The addition of salt to the protein in the most denaturing condition also reproduces the compaction effect, thereby supporting the role of anions in this phenomenon. Similar compaction of proteins is also observed in organic solvent/acid mixtures. The charge state of the compacted protein depends on the type of anions that formed ion pairs with a positive charge on the protein. The dissociation of ion pairs during the ionization process forms neutral acids that can be observed by HP-ESI using a soft ion introduction configuration.

Feedback Control of Electrospray with and without an External Liquid Pump Using the Spray Current and the Apex Angle of a Taylor Cone for ESI-MS.

An electrospray operated in the steady cone-jet mode is highly stable but the operating state can shift to pulsation or multijet modes owing to changes in flow rate, surface tension, and electrostatic variables. Here, a simple feedback control system was developed using the spray current and the apex angle of a Taylor cone to determine the error signal for correcting the emitter voltage. The system was applied to lock the cone-jet mode operation against external perturbations. For a pump-driven electrospray at a regulated flow rate, the apex angle of the Taylor cone decreased with increasing voltage. In contrast, for a voltage-driven electrospray with low flow resistance, the angle was found to increase with the emitter voltage. A simple algorithm based on iterative learning control was formulated and implemented using a personal computer to automatically correct the emitter voltage in response to the error signal. For voltage-driven electrospray ionization (ESI), the feedback control of the spray current can also be used to regulate the flow rate to an arbitrary value or pattern. Electrospray ionization-mass spectrometry (ESI-MS) with feedback control was demonstrated to produce ion signal acquisition with long-term stability that was insusceptible to the emulated external disturbances.

A Subtle Change in Nanoflow Rate Alters the Ionization Response As Revealed by Scanning Voltage ESI-MS.

The small charged droplet generated from the nanoelectrospray ionization (nanoESI) source at nL/min flow rate gives its unique feature of high-performance ionization. A continuous scan of the flow rate in this regime can trace the effect of droplet size in greater detail for a better understanding of the ionization process. To date, such practical implementation is hindered by the lack of a suitable liquid pump and the reproducibility of microcapillaries-based systems. Here, offline nanoESI mass spectrometry with a continuously varying flow rate in a dynamic range of several hundred pL/min to ∼100 nL/min was performed by the precision scanning of ESI high voltage (HV). The principle is based on the new paradigm of generating nanoelectrospray from a large Taylor cone with a known spray current-flow rate relationship. The instantaneous flow rate controlled by the HV was determined by simultaneous measurement of the spray current. The system is successfully applied to reveal the role of nanoflow rate on the average charge state of proteins, analysis of analyte mixture, and desalting effect. With the use of a buffer solution with high electric conductivity, a highly controllable oxidative modification was also observed by tuning the flow rate below a threshold of ∼5 nL/min, a finding that has potential application to on-demand oxygen labeling.

Hyphenation of high-temperature liquid chromatography with high-pressure electrospray ionization for subcritical water LC-ESI-MS.

High-pressure electrospray ionization (HP-ESI) performed under super-atmospheric pressure allows a stable and efficient electrospray of pure aqueous and/or superheated solutions even under a µL min-1 flow rate regime. In this paper, we report the direct coupling of the HP-ESI source to high-temperature liquid chromatography (HT-LC) operated at ≤30 µL min-1 flow rates. In addition to ESI, the ion source functions as a back-pressure regulator to keep the mobile phase in the liquid phase when the column is heated to >100 °C. Under an ion source pressure of 7 bar, the LC column can be operated up to 160 °C. LC is performed under isocratic elution, and besides the isothermal mode, the temperature of the column can also be programmed to increase the selectivity while keeping the ion source at a constant temperature. For a given solution flow rate, the analytical time can be shortened by increasing the column temperature. HT-LC-ESI-MS using pure water as the mobile phase with a capillary column is also demonstrated.

In vivo endoscopic mass spectrometry using a moving string sampling probe.

At present, endoscopy relies almost exclusively on optical microscopy and the accurate analysis such as MS interrogation is performed ex situ using biopsy. In this work, a novel probing system is developed to perform in situ and in vivo endoscopic mass spectrometry using a moving string for the sampling and transportation of material. A prototype of a mass spectrometric endoscope is constructed using an industrial endoscope and a commercial mass spectrometer. The sampling system consists of a moving cotton thread driven by motorized pulleys. When the target surface is touched by the sampling probe, the cotton thread "wipes" and transports the adhered sample to the ion source. Depending on the target analytes, desorption electrospray and atmospheric pressure chemical ionization sources are employed interchangeably for the desorption and ionization. The surface under analysis is not subjected to heat, organic solvents, high voltage or charged droplets. In situ endoscopic MS of a living mouse and surface analysis inside a volunteer subject's mouth are demonstrated.

Secondary ion yields for vacuum-type electrospray droplet beams measured with a triple focus time-of-flight analyzer.

RATIONALE: We previously developed a massive cluster ion beam gun for secondary ion mass spectrometry (SIMS) in which the primary beam source is a vacuum electrospray. The secondary ion yields produced by this method had not yet been measured with a commercial time-of-flight (TOF) secondary ion mass spectrometer, and the ionization performance was unknown. METHODS: A vacuum-type electrospray droplet ion gun was connected to a triple-focus TOF analyzer. The flight time of the secondary ions was measured using a sample-bias pulsing method, because a short pulse of the electrospray droplet beam could not be obtained. The secondary ion yields of an amino acid sample produced by the electrospray droplet beams and atomic Ga ion beams were compared. RESULTS: TOF secondary ion spectra were measured for the amino acid and peptide samples with a mass resolution of ~500 using the sample-bias pulsing method. The secondary ion yield of the amino acid sample produced with the 10 kV vacuum-type electrospray droplet beams was much higher than that produced by 10 kV Ga ion beams. In addition, the secondary ion yields for the peptide sample and amino acid samples were almost similar. CONCLUSIONS: This is the first report on secondary ion yields produced with vacuum-type electrospray droplet ion beams and measured with a semi-commercial TOF analyzer. The enhancement of secondary ion yields, in particular for relatively high-mass molecules, would be very useful in the SIMS analysis of a wide variety of biological samples. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of explosives using a hollow cathode discharge ion source.

RATIONALE: For public security and safety, it is highly desirable to develop an ion source for the detection of explosives that is highly sensitive, compact in size, robust, and does not use any special carrier gases such as helium. In this work, a hollow cathode discharge (HCD) ion source was developed for the detection of explosives using ambient air as a carrier gas. METHODS: To detect nonvolatile and thermally unstable explosives with high sensitivities, a new HCD ion source was designed and coupled with an ion trap mass spectrometer. RESULTS: Five explosives--hexamethylene triperoxide diamine (HMTD), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), pentaerythritol tetranitrate (PETN), nitroglycerin (NG) and trinitrotoluene (TNT)--were detected with limits of detection of lower than ng. The intensities of the NO3(-) adduct ions with RDX, PETN, and NG showed a marked increase with increase in ion source pressure in the range of 1-28 Torr. CONCLUSIONS: Because the major NOx(-) ions (x = 2, 3) produced in the plasma act as reagent ions in ion-molecule reactions of explosives, air is best suited as a carrier gas for the detection of explosives. It is proposed that the NOx(-) (x = 2, 3) and O3 contributed to the formation of [TNT-H](-) and [TNT-NO](-) ions, via the reactions NOx(-) + TNT → [TNT-H](-) + HNOx and [TNT](-) + O3 → [TNT-NO](-) + NO2 + O2.

Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure.

Nano electrospray with a solution flow rate below 100 nL min(-1) is usually performed using a metallized pulled glass capillary with an inner diameter tip of <10 µm. In this paper, we demonstrated that it was possible to achieve a stable solution flow rate of ~10 nL min(-1) for an aqueous solution using a disposable plastic pipette tip with an inner diameter of 100 µm as an ESI emitter. This pipette tip (gel loading tip) was originally designed for the loading of sample onto the gel in gel electrophoresis. The inner and outer diameters were much larger than those of a nanoESI emitter, but the low solution flow rate could be sustained by conducting the electrospray under a super-atmospheric gas pressure. The high pressure condition ensured the absence of electrical discharge which was essential for stable electrospray of aqueous solution. ESI with this low cost tip was equally as sensitive as the standard nanoESI emitter and capable of producing a long lasting signal from a small amount of sample liquid. Due to a small initial droplet size under the low flow rate regime, ESI with the present method exhibited the property of nanoESI where the ion suppression of the hydrophilic analyte by the hydrophobic compound present in the solution was greatly reduced. Furthermore, owing to its larger inner diameter, the tip relatively easily avoided the clogging problem often encountered when dealing with "dirty" samples when compared with a nanoESI emitter made from a fine glass capillary.

Piezoelectric inkjet assisted rapid electrospray ionization mass spectrometric analysis of metabolites in plant single cells via a direct sampling probe.

Direct sampling probe mass spectrometry (DSP-MS) enables fast and direct profiling of metabolites in biological samples. However, because the solvent amount for the online dissolution of acquired analytes is difficult to control, the detection sensitivity is not satisfactory. In this study, we present a modified version of the DSP-MS system for direct mass spectrometric profiling of metabolites in plant single cells. Two major improvements are introduced in this work, including a pointed-tip probe with high surface wetting properties, which is ten times finer than the previous version, and a piezoelectric inkjet system working as the auxiliary solvent delivery means. The probe tip can be controlled to insert into a cell through the cell wall. Metabolites loaded on the tip surface can be extracted by the auxiliary solvent and electrosprayed after applying a high direct current voltage. The unique features such as low cost, disposability and versatility make this technique a competitive tool for single cell analysis.

Direct ESI-MS of Ionic Liquids Using High-Pressure Electrospray From Large-Bore Emitters Made of Micropipette Tips.

Electrospray ionization mass spectrometry of neat undiluted ionic liquid (IL) and the analysis of protein with the doping of IL were performed using high-pressure electrospray. The use of disposable micropipette tips as emitters eased the handling of viscous and easy-to-clog samples and improved the reproducibility of the measurement. A high-pressure operation enabled the stable electrospray of the highly conductive IL from these relatively large bore emitters. The measurement of the current-voltage relationship of 1-ethyl-3-methylimidazolium tetrafluoroborate (Emim BF4) revealed an unusual negative differential resistance that has not been seen in the typical atmospheric or high-pressure electrospray. Mass spectrometric analysis of this IL also showed the characteristic response of various ion species with the emitter voltage. When added to the commonly used protein solution, the mass spectrum also showed protein peaks that correspond to the adduction of fluoroboric acid molecules (HBF4).

Real-time diagnosis of chemically induced hepatocellular carcinoma using a novel mass spectrometry-based technique.

Real-time analyses of hepatocellular carcinoma were performed in living mice to assess the applicability of probe electrospray ionization-mass spectrometry (PESI-MS) in medical diagnosis. The number of peaks and the abundance of ions corresponding to triacylglycerols (TAGs) were higher in cancerous tissues than in noncancerous tissues. Multiple sequential scans of the specimens were performed along a predetermined line extending over the noncancerous region to detect the boundary of the cancerous region. Our system successfully discriminated the noncancerous and cancerous tissues based on the intensities of the TAG ions. These results highlight the potential application of PESI-MS for clinical diagnosis in cancer.

Development of high-pressure probe electrospray ionization for aqueous solution.

RATIONALE: Probe electrospray ionization (PESI) is a recently developed method that uses a sharp solid needle as electrospray emitter and the sample is loaded to the needle tip by repetitive movement of the needle probe. This method has been previously used for the analysis of sample with high salt concentration and real-world samples without sample pretreatment. Although PESI is also applicable to aqueous solution, the ion signal stability and reproducibility were not satisfactory due to the spontaneous occurrence of corona discharge taking place on the metallic needle tip. METHODS: In the previous version of PESI, the ionization was performed under an open environment, thus ambient condition such as humidity and the presence of environmental contaminant also affected the performance of PESI. In this paper, we report a modified version of PESI in which the electrospray and the desolvation of charged droplets are performed inside an enclosed chamber which is pressurized to >1 atm with dry air. RESULTS: Under a super atmospheric condition, the dielectric strength of the ambient gas was enhanced and stable electrospray could to be initiated without the occurrence of corona discharge even for liquid sample with high surface tension such as pure water. A comparative study of atmospheric-pressure PESI and high-pressure PESI (HP-PESI) had been performed to demonstrate the improvement of PESI in sensitivity and signal stability for the detection analytes in aqueous solution. CONCLUSIONS: HP-PESI offers a higher sensitivity and signal stability over PESI due to the absence of gaseous breakdown, better desolvation, and higher ion sampling rate by the mass spectrometer.

Alternating current corona discharge/atmospheric pressure chemical ionization for mass spectrometry.

RATIONALE: Although alternating current (ac) corona discharge has been widely used in the fields of material science and technology, no reports have been published on its application to an atmospheric pressure chemical ionization (APCI) ion source. In this work, ac corona discharge for an APCI ion source has been examined for the first time. METHODS: The ambient atmospheric pressure ac corona discharge (15 kHz, 2.6 kVptp ) was generated by using a stainless steel acupuncture needle. The generated ions were measured using an ion trap mass spectrometer. A comparative study on ac and direct current (dc) corona APCI ion sources was carried out using triacetone triperoxide and trinitrotoluene as test samples. RESULTS: The ac corona discharge gave ion signals as strong as dc corona discharge for both positive and negative ion modes. In addition, softer ionization was obtained with ac corona discharge than with dc corona discharge. The erosion of the needle tip induced by ac corona was less than that obtained with positive mode dc corona. CONCLUSIONS: A good 'yardstick' for assessing ac corona is that it can be used for both positive and negative ion modes without changing the polarity of the high-voltage power supply. Thus, ac corona can be an alternative to conventional dc corona for APCI ion sources.

High pressure nanoelectrospray ionization mass spectrometry for analysis of aqueous solutions.

Nanoelectrospray ionization (nanoESI) with a very fine emitter and nanoliter solution flow rate is known to be suitable for aqueous solutions. However, under atmospheric pressure, its stability with aqueous solutions is not always guaranteed particularly in the negative ion mode where corona and arc discharge tend to occur more easily. Electrical discharge can be quenched to a certain extent by adding electron scavenging gases like SF6 or CO2 to the ion source. The onset potential that is required to induce the discharge also increases with an increase of gas pressure. Recently, we have reported on a series of high pressure electrospray ion sources that were stable in both positive and negative ion modes using air or N2 as the working gas. In this paper, we compare the performance of nanoelectrospray under atmospheric pressure and super-atmospheric pressure for the analysis of samples in aqueous solution. The comparative study was performed using the same ion source chamber that could be pressurized up to 6 bar. The pressure in the first pumping stage of the mass spectrometer was kept constant when the ion source pressure was changed by using an additional pump with variable pumping speed. High pressure nanoESI optimized at 2-3 bar demonstrated a 3-5 times improvement in ion signal intensity compared to atmospheric pressure nanoESI, and the signal stability was significantly improved particularly in the negative mode.

Tuning oxidative modification by a strong electric field using nanoESI of highly conductive solutions near the minimum flow rate.

Oxidative modification is usually used in mass spectrometry (MS) for labeling and structural analysis. Here we report a highly tunable oxidation that can be performed in line with the nanoESI-MS analysis at the same ESI emitter without the use of oxidative reagents such as ozone and H2O2, and UV activation. The method is based on the high-pressure nanoESI of a highly conductive (conductivity >3.8 S m-1) aqueous solution near the minimum flow rate. The ion source is operated under super-atmospheric pressure (0.5 MPa gauge pressure) to avoid the contribution of electric discharge. The analyte at the tip of the Taylor cone or in the emitter droplet can be locally oxidized in an on-demand manner by varying the nanoflow rate. With an offline nanoESI, the degree of oxidation, i.e., the average number of incorporated oxygen atoms, can be finely tuned by voltage modulation using spray current as the feedback signal. Oxidations of easily oxidized residues present in peptides/proteins and the double bonds of the unsaturated phosphatidylcholine occur at low flow rate operation (<5 nL min-1) when the electric field at the tip of the Taylor cone and the initially produced charged droplet reaches approximately 1.3 V nm-1. The oxidized ion signal responds instantaneously to changes in flow rate, indicating that the oxidation is highly localized. Using isotope labeling, it was found that the incorporated oxygen primarily originates from the gas phase, suggesting a direct oxidation pathway for the analyte enriched on the liquid surface via the reactive oxygen atoms formed by the strong electric field.

Bipolar Electrospray from Electrodeless Emitters for ESI without Electrochemical Reactions in the Sprayer.

A bipolar ESI source is developed to generate a simultaneous emission of charged liquid jets of opposite polarity from an electrodeless sprayer. The sprayer consists of two emitters, and the electrosprays are initiated by applying a high potential difference (HV) across the counter electrodes facing each emitter. The sprayer and the liquid delivery system are made of all insulators without metal components, thus enabling the total elimination of electrochemical reactions taking place at the liquid-electrode interface in the typical electrosprayer. The bipolar electrospray has been implemented using an online configuration that uses a syringe pump for flow rate regulation and an offline configuration that relies on HV for adjusting the flow rate. The voltage-current and flow rate-current relationships of bipolar electrospray were found to be similar to the standard electrospray. The application of bipolar ESI to the mass spectrometry of protein, peptide, and metallocene without electrochemically induced oxidation/reduction is demonstrated.

Vacuum electrospray of volatile liquids assisted by infrared laser irradiation.

RATIONALE: Current large cluster sources such as C(60) or argon utilize gas-phase sources which are of low-brightness and cannot be focused efficiently to better than 1 micron diameter spot size. The development of a high-brightness large cluster ion source is of critical importance to achieve high resolution in secondary ion mass spectrometry (SIMS) imaging of organics. METHODS: We propose a new high-brightness large cluster ion source, and a technique for producing a stable electrospray of volatile liquids under vacuum. It is known that vacuum electrospray of volatile liquids such as water is extremely difficult because of freezing of the liquids introduced in vacuum by evaporative cooling. To avoid freezing, the tip of the electrospray emitter was irradiated by a continuous wave infrared laser. RESULTS: Without continuous laser irradiation the vacuum electrospray of a water/methanol solution was unstable with respect to the shapes of the Taylor cone and current, whereas continuous laser irradiation produced a stable electrospray of water. The typical modes of electrospray were clearly observed with an optical microscope even under vacuum conditions. A stable vacuum electrospray could be achieved by improving the vacuum pressure to suppress electric discharge and by using the laser to maintain the liquid state. CONCLUSIONS: This is the first description of the production of a stable vacuum electrospray of volatile liquids such as water. This vacuum electrospray technique can be expected to produce a novel high-brightness large cluster ion beam source.