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.

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.

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.

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.

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.

Remote sampling mass spectrometry for dry samples: Sheath-flow probe electrospray ionization (PESI) using a gel-loading tip inserted with an acupuncture needle.

RATIONALE: Probe electrospray ionization (PESI) is only applicable to liquid or wet samples. In this study, a sheath-flow PESI method for remote sampling mass spectrometry that can be applied to dry samples was developed. METHODS: An acupuncture needle (0.12 mm outer diameter, 700 nm tip diameter) was inserted into a gel-loading tip with a 0.1 mm protrusion out of the tip. Analytes were extracted by filling the latter tip with solvent and softly touching the sample surface for a short time (<1 s). A high voltage was applied to the acupuncture needle, and mass spectra of analytes were obtained by self-aspirating electrospray. RESULTS: Dry samples, such as lines of ballpoint pen ink on paper, pharmaceutical tablets, instant coffee, brown rice, and narcotics, gave strong ion signals. The sample carryover was negligible. The sequential electrospray was observed to be similar to conventional PESI. The limits of detection (LODs) for morphine and rhodamine B were found to be of the order of picograms. CONCLUSIONS: Because of its simplicity and versatility, sheath-flow PESI is a promising technique for on-site and nondestructive profile analysis of dry samples with bulky and complicated shapes, with a spatial resolution of ~0.3 mm.

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.

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.

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.

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.

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.

Solid probe assisted nanoelectrospray ionization mass spectrometry for biological tissue diagnostics.

To perform remote and direct sampling for mass spectrometry, solid probe assisted nanoelectrospray ionization (SPA-nanoESI) has been newly developed. After capturing the sample on the tip of the needle by sticking it to the biological tissue, the needle was inserted into the solvent-preloaded nanoESI capillary from the backside. NanoESI gave abundant ion signals for human kidney tissues and the liver of a living mouse. The method is easy to operate and versatile because any biological specimen could be sampled away from the mass spectrometer. Minimal invasiveness is another merit of this method.

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.

Development of double cylindrical dielectric barrier discharge ion source.

This paper deals with the dielectric barrier discharge (DBD) ion source composed of the outer cylindrical dielectric tube and the inner grounded metallic tube electrode. The sample gas is supplied through the inner ceramic tube. In this ion source, the DBD plasma is localized in the DBD tube so that the sample gases can be ionized just outside of the ceramic tube by the DBD excited helium gas without being exposed in the plasma jet. Besides, ambient air does not take part in the ionization of the sample vapor because ionization takes place inside the DBD ion source. Thus, this method is totally free from contaminants in ambient air. It was found that this ion source is capable of soft, high-sensitivity, and reproducible ionization. Application of this technique to the analysis of methamphetamine, carbaryl and basil leaf was given.

Miniaturized String Sampling Probe and Electrospray Extraction/Ionization within the Ion Inlet Tube for Mass Spectrometric Endoscopy.

A moving string sampling probe and a new ESI based ionization source that can be readily incorporated into the existing endoscopes are developed for performing in vivo mass spectrometry during the endoscopic procedure. The medical-grade silk suture driven by a stepping motor is used to perform the sampling on the region of interest when the probe head is brought gently into contact with the surface of the gastrointestinal tissue. The tissues and the compounds adhered to the sampling string are transported to an ionization region inside the ion inlet tube in which they are extracted and ionized by the charging droplets generated from an electrospray outside the ion inlet. Since the extraction/ionization and sampling processes are isolated, organic solvents, high voltage (HV), and heating can be used for the optimization of ionization without compromising the biocompatibility of the sampling probe. The demonstration of the in vivo analysis of the gastric mucosa of a mouse is performed using a 2 m long gastrointestinal endoscope.

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.

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.

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).