Solvent effect on the detection of peptides and proteins by nanoelectrospray ionization mass spectrometry: Anomalous behavior of aqueous 2-propanol.

To investigate the solvent effect on the detection of peptides and proteins, nanoelectrospray mass spectra were measured for mixtures of 1 % acetic acid and 5 × 10-6 M gramicidin S (G), ubiquitin (U), and cytochrome c (C) in water (W), methanol (MeOH), 1-propanol (1-PrOH), acetonitrile (AcN), and 2-propanol (2-PrOH). Although doubly protonated G (G2+) and multiply protonated U (Un+) and C (Cn+) were readily detected with a wide range of mixing ratios of W solutions for MeOH, 1-PrOH, and AcN, Cn+ was totally suppressed for the solutions with mixing ratios (v/v) of W/2-PrOH (50/50) and (70/30). However, denatured Cn+ started to be detected with W/2-PrOH (90/10) together with Gn+ (n = 1, 2) and native Un+ (n = 6-8). At the mixing ratio of W/2-PrOH (95/5), native Cn+ (n = 7-10) together with Gn+ (n = 1, 2) and native Un+ (n = 6-8) were detected with high ion intensities. The use of W/2-PrOH (95/5) is profitable because it enables the detection of native proteins with high detection sensitivities.

Analysis of human skin sebum and animal meats by heat pulse desorption/mass spectrometry using proximity corona discharge ionization.

Recently, we have developed heat pulse desorption/mass spectrometry (HPD/MS). In HPD/MS, a heated N2 gas pulse was directed to the sample surface and desorbed analytes were mass analyzed by corona discharge ionization/mass spectrometry using an Orbitrap mass spectrometer. In this work, HPD/MS was applied to the analysis of skin surface components sampled from the forehead, nose, and jaw of three volunteers. It was found that various kinds of biological compounds such as squalene, free fatty acids, wax esters, triacylglycerols, and amino acids were detected. The simultaneous detection of compounds with a wide range of proton affinities suggests that the occurrence of consecutive proton transfer reactions is less likely to occur in the present experimental system. This is mainly due to the short distance of 1.5 mm between the tip of the corona needle and the inlet of the mass spectrometer (i.e., proximity corona discharge ion source). Under this condition, the transition time of the primary reactant ions (e.g., H3O+) from the tip of the corona discharge needle to the ion sampling orifice is roughly estimated to be ∼20 µs. This value nearly corresponds to the reaction lifetime of exoergic proton transfer reactions with a rate constant: ∼10-9 cm3 s-1 for the analytes of 1 ppm. Accordingly, analytes with concentrations less than 1 ppm would be ionized semi-quantitatively by the present method, making this method highly suitable for the rapid analysis of samples composed of complex mixture of compounds, e.g., non-target lipidomics.

An Investigation of the Non-selective Etching of Synthetic Polymers by Electrospray Droplet Impact/Secondary Ion Mass Spectrometry (EDI/SIMS).

Among the various types of cluster secondary ion mass spectrometry (SIMS), electrospray droplet impact/secondary ion mass spectrometry (EDI/SIMS) is unique due to its high ionization efficiency and non-selective atomic/molecular-level surface etching ability. In this study, EDI/SIMS was applied to the non-selective etching of synthetic polymers of polystyrene (PS) and poly(9,9-di-n-octylfluonyl-2,7diyl) (PFO) deposited on a silicon substrate. The polymers gave characteristic fragment ions and the mass spectra remained unchanged with prolonged EDI irradiation time, indicating that non-selective etching can be achieved by EDI irradiation, a finding that is consistent with our previous reports based on EDI/X-ray photoelectron spectroscopy analyses. From the irradiation time and film thickness, the etching rates for PS and PFO were roughly estimated to be 0.6 nm/min and 0.15 nm/min, respectively, under the experimental conditions that were used. After the depletion of polymer sample on the surface, ion signals originating from the exposed silicon substrate were observed. This indicates that EDI/SIMS is applicable to the analysis of the interface of multilayered films composed of organic and inorganic materials.

Heat Pulse Desorption of Low-Volatility Compounds by a Heated N2 Gas Pulse with Mass Spectrometry.

For the thermal desorption of low-volatility compounds, rapid heating followed by instant cooling is desirable to suppress thermal decomposition. In this work, a new thermal desorption method, heat pulse desorption (HPD), was developed. A heated N2 gas pulse (350 °C, 50 ms) was directed to the solid sample surface, and desorbed analytes were ionized by DC corona discharge and mass analyzed by an Orbitrap mass spectrometer. Because heat transfer from the heated N2 gas to the solid surface is not very efficient, desorption of the solid sample occurs at a certain temperature before reaching 350 °C. In short, there is a self-controlling desorption depending on the volatility of each analyte. Because the exit of the copper tube for gas blowing is separated from the sample surface, no carryover occurs, enabling the repetitive analysis of samples. HPD was applied to various compounds such as narcotics, pharmaceutical tablets, and explosives. Because analysis is completed within a few seconds per sample, this method is highly useful for quick and consecutive analysis of real samples, having potential utility in food quality control, counterfeit drugs analysis, and the detection of explosives for safety and security.

Corona Discharge and Field Electron Emission in Ambient Air Using a Sharp Metal Needle: Formation and Reactivity of CO3 -• and O2 -•.

CO3 -• and O2 -• are known to be strong oxidizing reagents in biological systems. CO3 -• in particular can cause serious damage to DNA and proteins by H• abstraction reactions. However, H• abstraction of CO3 -• in the gas phase has not yet been reported. In this work we report on gas-phase ion/molecule reactions of CO3 -• and O2 -• with various molecules. CO3 -• was generated by the corona discharge of an O2 reagent gas using a cylindrical tube ion source. O2 -• was generated by the application of a 15 kHz high frequency voltage to a sharp needle in ambient air at the threshold voltage for the appearance of an ion signal. In the reactions of CO3 -•, a decrease in signal intensities of CO3 -• accompanied by the simultaneous increase of that of HCO3 - was observed when organic compounds with H-C bond energies lower than ∼100 kcal mol-1 such as n-hexane, cyclohexane, methanol, ethanol, 1-propanol, 2-propanol, and toluene were introduced into the ion source. This clearly indicates the occurrence of H• abstraction. O2 -• abstracts H+ from acid molecules such as formic, acetic, trifluoroacetic, nitric and amino acids. Gas-phase CO3 -• may play a role as a strong oxidizing reagent as it does in the condensed phase. The major discharge product CO3 -• in addition to O2 -•, O3, and NO x • that are formed in ambient air may cause damage to biological systems.

Probe Electrospray Ionization (PESI) and Its Modified Versions: Dipping PESI (dPESI), Sheath-Flow PESI (sfPESI) and Adjustable sfPESI (ad-sfPESI).

In 2007, probe electrospray ionization/mass spectrometry (PESI/MS) was developed. In this technique, the needle is moved down along a vertical axis and the tip of the needle touched to the sample. After capturing the sample at the needle tip, the needle is then moved up and a high voltage is applied to the needle at the highest position to generate electrospray. Due to the discontinuous sampling followed by the generation of spontaneous electrospray, sequential and exhaustive electrospray takes place depending on the surface activity of the analytes. As modified versions of PESI, dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI) have been developed. These methods are complementary to each other and they can be applicable to surface and bulk analysis of various biological samples. In this article, the characteristics of these methods and their applications to real samples will be reviewed.

Flash desorption of low-volatility compounds deposited on a heated solid substrate (90°C) by dripping liquid methanol.

RATIONALE: Desorption of low-volatility compounds deposited on a solid substrate by dripping a methanol drop was explored. METHODS: Low-volatility compounds such as drugs and explosives were deposited/dried on the substrate at 35°C. After increasing the temperature to 90°C, 5 µL methanol was dripped onto the substrate. The desorbed analytes were ionized by alternating current corona discharge and analyzed by mass spectrometry. RESULTS: Flash desorption for drugs and explosives was observed accompanied by the rapid evaporation of methanol. However, saccharides, fullerene, cholesterol, and gramicidin S were not detected by the present method. CONCLUSIONS: It was suggested that surface-active compounds were desorbed at the peripheral front region of the spreading liquid methanol accompanied by rapid evaporation of methanol.

Robotic sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS): development of a touch sensor for samples in a multiwell plastic plate.

In the previous work, sheath-flow probe electrospray ionization (sfPESI) equipped with a touch sensor was developed for conducting samples. In this work, a capacitiance-sensitive touch sensor that can be applicable to samples prepared in a nonconducting plastic multiwell plate was developed. The radiofrequency with 5 kHz and 4.5 Vpp was applied to the metal substrate on which the plastic plate was placed. The probe tip stopped at the position where it touched the surface of the liquid solution prepared in the plastic multiwell plate by detecting the displacement current flowing through the capacitance of the circuit. By coupling a nondisposable sfPESI probe with a table-top 3-axis robot, consecutive analysis of peptides, proteins, drugs, and real samples was performed. The carry-over by the consecutive analyses was suppressed to minimal by cleansing the probe tip using the solvent of water/methanol/acetonitrile (1/1/1).

On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice.

Although a loss of healthy pollen grains induced by metabolic heat responses has been indicated to be a major cause of heat-induced spikelet sterility under global climate change, to date detailed information at pollen level has been lacking due to the technical limitations. In this study, we used picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS) to directly determine the metabolites in heat-treated single mature pollen grains in two cultivars, heat-tolerant cultivar, N22 and heat-sensitive cultivar, Koshihikari. Heat-induced spikelet fertility in N22 and Koshihikari was 90.0% and 46.8%, respectively. While no treatment difference in in vitro pollen viability was observed in each cultivar, contrasting varietal differences in phosphatidylinositol (PI)(34:3) have been detected in mature pollen, together with other 106 metabolites. Greater PI content was detected in N22 pollen regardless of the treatment, but not for Koshihikari pollen. In contrast, there was little detection for phosphoinositide in the single mature pollen grains in both cultivars. Our findings indicate that picoPPESI-MS analysis can efficiently identify the metabolites in intact single pollen. Since PI is a precursor of phosphoinositide that induces multiple signaling for pollen germination and tube growth, the active synthesis of PI(34:3) prior to germination may be closely associated with sustaining spikelet fertility even at high temperatures.

Pulsed Nano-Electrospray Ionization with a High Voltage (4000 V) Pulse Applied to Solutions in the Range of 200 ns to 1 ms.

To initiate electrospray, it is necessary to accumulate excess charges on the liquid surface until the outward electrostatic pressure (PE) overwhelms the inward pressure Pγ originating from the surface tension of the liquid droplet. In this report, electrospray mass spectrometry for solutions of gramicidin S (G), ubiquitin (U), and cytochrome c (C) in H2O/CH3OH (1/1) was performed as a function of the high voltage (4000 V) pulse width in the range of 200 ns to 1 ms using a 4 µm i.d. glass capillary. Multiply protonated ions for all three samples started to be detected with the 300 µs pulse width. Denaturation of C and U proceeded with an increase of the pulse width. When the bias voltage of ∼880 V that was lower than the threshold voltage for the generation of continuous electrospray (∼1000 V) was applied to the solution, multiply protonated G, U, and C were detected with the 200 ns pulse width. After the depletion of excess charges in 200 ns, it took 10-100 µs to regenerate electrospray.

Rapid desorption of low-volatility compounds in liquid droplets accompanied by the flash evaporation of solvent below the Leidenfrost temperature.

RATIONALE: The objective of this work is to study the interaction of methanol droplets with the heated surface for the improved detection of low-volatility and thermally labile compounds by the flash evaporation that occurs below the Leidenfrost temperature. METHODS: 5 µL solutions of low-volatility compounds in methanol were introduced into the heated tube. Desorbed analytes were ionized in the sealed atmospheric pressure chemical ionization (APCI) source by direct current (DC) corona discharge using air as the reagent gas. RESULTS: The rapid desorption of low-volatility compounds accompanied by the flash evaporation of methanol solvent was observed in the temperature range of 60-100°C. Linear relationships between the signal intensities and the solute concentrations in the range of 0.01-5 ppm for morphine, cocaine, methamphetamine, and amphetamine were obtained at 95°C. CONCLUSIONS: The observed rapid desorption of low-volatility compounds below the Leidenfrost temperature would provide useful information in many fields, e.g., the interaction of liquid droplets with heated matter, liquid sample introduction into the injection port of a gas chromatograph, coupling of the flash evaporation with pulse valve operated miniaturized mass spectrometer, etc.

Point Analysis of Foods by Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry (sfPESI/MS) Coupled with a Touch Sensor.

For quick, noninvasive, and high-sensitivity surface analysis of foods and agricultural products, a touch sensor was developed and applied to sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS). Upon making contact with the sample, the probe stopped by detecting the current flowing through the circuit and analytes on the sample surface were extracted in the solvent preloaded in the plastic capillary. By lifting up the probe to the default position, an electrospray ionization mass spectrum of the sample was obtained. By scanning the sample stage using a programming tool, a point analysis of targeted positions of biological samples with a spot diameter of ≤0.3 mm was achieved. It took less than 10 s for one sample spot. This method was applied to various plants and animal tissues.

Real-time analysis of living animals and rapid screening of human fluid samples using remote sampling electrospray ionization mass spectrometry.

Real-time and in-situ mass-spectrometry analyses of living animal and biological sample were performed using a novel remote sampling electrospray ionization (RS-ESI) probe. Unlike conventional ESI, in which injection or syringe loading is required for sample introduction, the RS-ESI probe ionizes the samples when the sampling capillary is in contact with the sample. As the sampling capillary is electrically held at ground potential, the safety of the animal and operator is assured. The liquid sample is aspirated to the ESI emitter at the other end of the capillary by the Venturi effect. Subsequently, the electrospray is generated when a high voltage is applied to the counter electrode placed inside the ion source chamber. The probe unit is attached to the mass spectrometer with a long flexible tube and its position can be freely manipulated during the analysis. In this report, we demonstrate a real-time analysis of a living mouse liver and an automatic analysis of 138 serum samples using this new technique.

Component Profiling in Agricultural Applications Using an Adjustable Acupuncture Needle for Sheath-Flow Probe Electrospray Ionization/Mass Spectrometry.

In previous work, probe electrospray ionization/mass spectrometry (PESI/MS) and sheath-flow probe electrospray ionization/mass spectrometry (sfPESI/MS) were reported for the rapid and minimally invasive analysis of food. In this work, a modified version of sfPESI will be reported. The sample surface was pricked with an acupuncture needle inserted in the sfPESI probe that protruded from the terminus of the tip by 5 mm. The invasion depth of the needle into the sample was ∼1 mm. After sampling, the needle was retracted into the solvent-preloaded capillary with a protrusion length of 0.1-0.2 mm from the tip. A mass spectrum of the sample captured on the needle was obtained by applying a high voltage to the needle. This method could be applicable to profiling analyses of plants with the epicuticular wax covering on the surfaces that are difficult to analyze by sf-PESI. The on-site mass spectrometric analysis for a growing apricot in the field was performed to monitor the developing stage of the fruit.

Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm.

Heat-induced chalkiness of rice grains is a major concern for rice production, particularly with respect to climate change. Although the formation of chalkiness in the endosperm is suppressed by nitrogen, little is known about the cell-specific dynamics of this process. Here, using picolitre pressure-probe electrospray-ionization mass spectrometry together with transmission electron microscopy and turgor measurements, we examine heat-induced chalkiness in single endosperm cells of intact rice seeds produced under controlled environmental conditions. Exposure to heat stress decreased turgor pressure and increased the cytosolic accumulation of sugars, glutathione, and amino acids, particularly cysteine. Heat stress also led to a significant enlargement of the protein storage vacuoles but with little accumulation of storage proteins. Crucially, this heat-induced partial arrest of amyloplast development led to formation of chalkiness. Whilst increased nitrogen availability also resulted in increased accumulation of amino acids, there was no decrease in turgor pressure. The heat-induced accumulation of cysteine and glutathione was much less marked in the presence of nitrogen, and storage proteins were produced without chalkiness. These data provide important information on the cell dynamics of heat acclimation that underpin the formation of chalkiness in the rice endosperm. We conclude that rice seeds employ multiple strategies to mitigate the adverse effects of heat stress in a manner that is dependent on nitrogen availability, and that the regulation of protein synthesis may play a crucial role in optimizing organelle compartmentation during heat adaption.

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.

Electrospray Generated from the Tip-Sealed Fine Glass Capillary Inserted with an Acupuncture Needle Electrode.

In electrospray, excess charges are supplied to a sample solution by the occurrence of electrochemical reactions. Recently, different versions of electrospray, e.g., dielectric barrier electrospray ionization, inductive desorption electrospray ionization, and electrostatic-ionization driven by dielectric polarization, have been reported in which the sample solution was not in direct contact with the metal electrode but separated by dielectric materials. The objective of the current work is to elucidate the mechanism of dielectric barrier electrospray. A sealed borosilicate glass capillary inserted with a fine acupuncture needle was used as a probe. A sample solution (~ 400 nL) was captured on the glass capillary tip and a positive high voltage (HV) pulse (+ 4.5 kV) was applied to the internal metal electrode. Mass spectra were measured as a function of the HV pulse width from µs to 10 s. Ions started to be detected with the pulse width of ~ 5 ms. The ion intensities increased slowly with time and reached a plateau in a few seconds. The charge distribution of cytochrome c [M + nH]n+ shifted to higher n values from a few ms to seconds. In addition to cone-jet mode normal electrospray that lasted until all the liquid sample was depleted from the glass tip, the polarization-induced electrospray ionization was observed at the early stage of the HV application. Graphical Abstract ᅟ.

Development of a Vacuum Electrospray Droplet Ion Gun for Secondary Ion Mass Spectrometry.

Atmospheric pressure electrospray had been used in previous studies to generate massive water droplet ion beams, and the beams successfully achieved efficient desorption/ionization of biomolecules, low damage etching of polymers and nonselective etching of metal oxides. However, this droplet ion beam was not practical as a primary ion beam for surface analysis instruments because it required differential pumping and lacked adequate beam current and density. To improve the beam performance, we have proposed to use vacuum electrospray of aqueous solutions as a beam source, and developed a technique for producing a stable electrospray of aqueous solution in vacuum. We also designed a prototype of a vacuum electrospray droplet ion gun, and measured the beam properties. Finally, the applicability of this ion gun in secondary ion mass spectrometry is discussed.

Evidence for preservation of vacuolar compartments during foehn-induced chalky ring formation of Oryza sativa L.

MAIN CONCLUSION: Vacuolar compartments being sustained among the amyloplasts inadequately accumulated in rice endosperm cells are the main cause of chalky ring formation under dry wind conditions. Foehn-induced dry wind during the grain-filling stage induces shoot water deficit in rice (Oryza sativa L.) plants, which form a ring-shaped chalkiness in their endosperm that degrades milling quality and rice appearance. Air spaces formed in several inner cells cause significant transparency loss due to irregular light reflection. Although starch synthesis was suggested to be retarded by osmotic adjustment at foehn-induced moderately low water potential, the source of these air spaces remains unknown. We hypothesised that the preservation of vacuoles accompanied by a temporary reduction in starch biosynthesis in the inner cells leads to the chalky ring formation. Panicle water status measurement, light and transmission electron microscopic (TEM) observations, and an absolute qPCR analysis were conducted. Most starch synthesis-related genes exhibited temporarily reduced expression in the inner zone in accordance with the decrease in panicle water status. TEM observations provided evidence that vacuolar compartments remained among the loosely packed starch granules in the inner endosperm cells, where a chalky ring appeared after kernel dehydration. Taken together, we propose that vacuolar compartments sustained among the amyloplasts inadequately accumulated in rice endosperm cells and caused air space formation that leads to ring-shaped chalkiness under dry wind conditions.