Desorption in Mass Spectrometry.

In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

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 analysis of anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption-dielectric barrier discharge ionization mass spectrometry.

Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the consumption of performance enhancing anabolic steroids by athletes. The present article describes a novel strategy for identifying the trace anabolic steroids in urine using Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are enriched within a liquid droplet during the thermal desorption process and desorbed all-together at the last moment of droplet evaporation in a short time domain. The desorbed molecules were ionized using a dielectric barrier discharge ion-source in front of the mass spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement with several orders of magnitude compared to the thermal desorption at a lower temperature. The limits of detection (LODs) of various steroid molecules were found to be in the range of 0.05-0.1 ng mL(-1) for standard solutions and around two orders of magnitude higher for synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by using a simple and rapid dichloromethane extraction technique. The analytical figures of merit of this technique were evaluated at open atmosphere using suitable internal standards. The technique is simple and rapid for high sensitivity and high throughput screening of anabolic steroids in urine.

Biomolecular analysis and biological tissue diagnostics by electrospray ionization with a metal wire inserted gel-loading tip.

A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (~100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid was sampled from the biological tissue by a needle and was inserted into the solvent-preloaded gel-loading tip). Selective detection of lipids and proteins, such α and ß chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

Trace level detection of explosives in solution using leidenfrost phenomenon assisted thermal desorption ambient mass spectrometry.

The present paper demonstrates the detection of explosives in solution using thermal desorption technique at a temperature higher than Leidenfrost temperature of the solvent in combination with low temperature plasma (LTP) ionization. Leidenfrost temperature of a solvent is the temperature above which the solvent droplet starts levitation instead of splashing when placed on a hot metallic surface. During this desorption process, slow and gentle solvent evaporation takes place, which leads to the pre-concentration of less-volatile explosive molecules in the droplet and the explosive molecules are released at the last moment of droplet evaporation. The limits of detection for explosives studied by using this thermal desorption LTP ionization method varied in a range of 1 to 10 parts per billion (ppb) using a droplet volume of 20 µL (absolute sample amount 90-630 fmol). As LTP ionization method was applied and ion-molecule reactions took place in ambient atmosphere, various ion-molecule adduct species like [M+NO2](-), [M+NO3](-), [M+HCO3](-), [M+HCO4](-) were generated together with [M-H](-) peak. Each peak was unambiguously identified using 'Exactive Orbitrap' mass spectrometer in negative ionization mode within 3 ppm deviation compared to its exact mass. This newly developed technique was successfully applied to detect four explosives contained in the pond water and soil sample with minor sample pre-treatment and the explosives were detected with ppb levels. The present method is simple, rapid and can detect trace levels of explosives with high specificity from solutions.

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.

Development of sheath-flow probe electrospray ionization (SF-PESI).

Probe electrospray ionization (PESI) uses a sharp solid needle as electrospray emitter. This method was found to be applicable to the analysis of real-world samples with high concentrations of salts and detergents without sample pretreatment. Since PESI is only applicable to wet samples but not to dry samples, sheath-flow PESI (SF-PESI) has been developed. The metal needle was inserted into the fine plastic capillary with a protrusion of 0.1-0.2 mm from the capillary terminus. The solvent was supplied continuously through the capillary. At the lowest position of the probe, solvent flowing out from the capillary makes the sample wet and extracts the analytes from the surface. The extracted analytes were electrosprayed at the highest position of the needle. SF-PESI was successfully applied to samples such as narcotics, tablets, bill, fruits, potatoes, etc.

Direct electrospray ionization mass spectrometric profiling of real-world samples via a solid sampling probe.

This study presents a novel direct analysis strategy for rapid mass spectrometric profiling of biochemicals in real-world samples via a direct sampling probe (DSP) without sample pretreatments. Chemical modification is applied to a disposable stainless steel acupuncture needle to enhance its surface area and hydrophilicity. After insertion into real-world samples, biofluid can be attached on the DSP surface. With the presence of a high DC voltage and solvent vapor condensing on the tip of the DSP, analyte can be dissolved and electrosprayed. The simplicity in design, versatility in application aspects, and other advantages such as low cost and disposability make this new method a competitive tool for direct analysis of real-world samples.

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 sheath-flow probe electrospray ionization mass spectrometry and its application to real time pesticide analysis.

For the real time and direct analysis of chemical constituents from living beings and dry sample, sheath-flow probe electrospray ionization mass spectrometry (SF-PESI-MS) has been newly developed. The components from dry or semidry biological tissues can be extracted using the solvent and picked up by the needle for electrospray. This technique was applied to real-time pesticide analysis of living plants. The results have been validated with that of a well-known system, liquid extraction surface analysis mass spectrometry (LESA-MS). It is demonstrated that SF-PESI-MS can produce reasonable ionization efficiency, which is confirmed by LESA-MS.

Leidenfrost phenomenon-assisted thermal desorption (LPTD) and its application to open ion sources at atmospheric pressure mass spectrometry.

This work describes the development and application of a new thermal desorption technique that makes use of the Leidenfrost phenomenon in open ion sources at atmospheric pressure for direct mass spectrometric detection of ultratrace levels of illicit, therapeutic, and stimulant drugs, toxicants, and peptides (molecular weight above 1 kDa) in their unaltered state from complex real world samples without or with minor sample pretreatment. A low temperature dielectric barrier discharge ion source was used throughout the experiments and the analytical figures of merit of this technique were investigated. Further, this desorption technique coupled with other ionization sources such as electrospray ionization (ESI) and dc corona discharge atmospheric pressure chemical ionization (APCI) in open atmosphere was also investigated. The use of the high-resolution 'Exactive Orbitrap' mass spectrometer provided unambiguous identification of trace levels of the targeted compounds from complex mixtures and background noise; the limits of detection for various small organic molecules and peptides treated with this technique were at the level of parts per trillion and 10(-9) M, respectively. The high sensitivity of the present technique is attributed to the spontaneous enrichment of analyte molecules during the slow evaporation of the solvent, as well as to the sequential desorption of molecules from complex mixtures based on their volatilities. This newly developed desorption technique is simple and fast, while molecular ions are observed as the major ions.

Biomolecular analysis and cancer diagnostics by negative mode probe electrospray ionization.

We have examined several combinations of solvents and probes with the aim of optimizing the ionization conditions for biomolecules e.g., proteins, peptides and lipids by negative mode probe electrospray ionization mass spectrometry (PESI-MS). With the data presented in this study, negative-mode PESI-MS can be considered as a potential tool for biomolecular analysis and cancer diagnostics because of its simplicity in instrumental configuration. A sharper sampling probe was found to be better for obtaining high quality mass spectra because it can generate stable electrospray without the occurrence of gas breakdown. Although the best conditions may depend on each sample, aqueous organic solvent solutions, especially isopropanol-H(2)O (1/1) with a pH of ≥7, are shown to be preferable for negative-mode PESI-MS, which was successfully applied to colon cancer diagnosis.

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.

Application of probe electrospray ionization mass spectrometry (PESI-MS) to clinical diagnosis: solvent effect on lipid analysis.

We have examined several combinations of solvents with the aim of optimizing the ionization conditions for molecular diagnosis of malignant tumours by PESI-MS. Although the best conditions may depend on the actual species in the sample, the optimal conditions for renal cell carcinoma (RCC) were achieved by using alcohols. PESI-MS successfully delineated the differential expression of phospholipids (PCs) and triacylglycerols (TAGs) in noncancerous and RCC tissues by using these solvent systems. This study paves the way for the application of PESI-MS in medical samples.

Analysis of renal cell carcinoma as a first step for developing mass spectrometry-based diagnostics.

Immediate diagnosis of human specimen is an essential prerequisites in medical routines. This study aimed to establish a novel cancer diagnostics system based on probe electrospray ionization-mass spectrometry (PESI-MS) combined with statistical data processing. PESI-MS uses a very fine acupuncture needle as a probe for sampling as well as for ionization. To demonstrate the applicability of PESI-MS for cancer diagnosis, we analyzed nine cases of clear cell renal cell carcinoma (ccRCC) by PESI-MS and processed the data by principal components analysis (PCA). Our system successfully delineated the differences in lipid composition between non-cancerous and cancerous regions. In this case, triacylglycerol (TAG) was reproducibly detected in the cancerous tissue of nine different individuals, the result being consistent with well-known profiles of ccRCC. Moreover, this system enabled us to detect the boundaries of cancerous regions based on the expression of TAG. These results strongly suggest that PESI-MS will be applicable to cancer diagnosis, especially when the number of data is augmented.

Mass spectrometry of rhenium complexes: a comparative study by using LDI-MS, MALDI-MS, PESI-MS and ESI-MS.

A group of rhenium (I) complexes including in their structure ligands such as CF(3)SO(3)-, CH(3)CO(2)-, CO, 2,2'-bipyridine, dipyridil[3,2-a:2'3'-c]phenazine, naphthalene-2-carboxylate, anthracene-9-carboxylate, pyrene-1-carboxylate and 1,10-phenanthroline have been studied for the first time by mass spectrometry. The probe electrospray ionization (PESI) is a technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid metal needle. In this work, mass spectra for organometallic complexes obtained by PESI were compared with those obtained by classical ESI and high flow rate electrospray ionization assisted by corona discharge (HF-ESI-CD), an ideal method to avoid decomposition of the complexes and to induce their oxidation to yield intact molecular cation radicals in gas state [M](+·) and to produce their reduction yielding the gas species [M](-·). It was found that both techniques showed in general the intact molecular ions of the organometallics studied and provided additional structure characteristic diagnostic fragments. As the rhenium complexes studied in the present work showed strong absorption in the UV-visible region, particularly at 355 nm, laser desorption ionization (LDI) mass spectrometry experiments could be conducted. Although intact molecular ions could be detected in a few cases, LDI mass spectra showed diagnostic fragments for characterization of the complexes structure. Furthermore, matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained. Nor-harmane, a compound with basic character, was used as matrix, and the intact molecular ions were detected in two examples, in negative ion mode as the [M](-·) species. Results obtained with 2-[(2E)-3-(4-tert-buthylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) as matrix are also described. LDI experiments provided more information about the rhenium complex structures than did the MALDI ones.

Online electrospray ionization mass spectrometric monitoring of protease-catalyzed reactions in real time.

Although there are a lot of well established methods for monitoring enzyme-catalyzed reactions, most of them are based on changes in spectroscopic properties during the conversion of substrates to products. However, reactions without optical changes are common, which are inapplicable to these spectroscopic methods. As an alternative technique for enzymologic research, mass spectrometry (MS) is favored due to its specificity, sensitivity, and the ability to obtain stoichiometric information. In this work, probe electrospray ionization (PESI) source coupled with a time of flight mass spectrometer was employed to monitor some typical protease-catalyzed reactions, including pepsinolysis and trypsinolysis of cytochrome c in real time. Due to the high electrical conductivity of each reaction system, corona discharges are likely to occur, which would decrease intensities of mass spectrometric signals. An ultra-fine sampling probe and an auxiliary vapor spray were adopted to prevent corona discharges. Experimental results from peptic and tryptic digestions of cytochrome c showed different and characteristic catalytic pathways. With the data presented in this study, PESI-MS can be considered as a potential tool for real-time monitoring of enzymatic reactions because of its simplicity in instrumental configuration, wide applicability under harsh conditions, and flexibility in combination with other techniques.

Detection of protein from detergent solutions by probe electrospray ionization mass spectrometry (PESI-MS).

Detergents are necessarily used for different extraction protocols of proteins from biological cells or tissues. After the extraction, elimination of detergent is necessary for the better performance of electrospray ionization mass spectrometry (ESI-MS). Elimination of detergents is laborious and time-consuming, and also sample loss may be unavoidable. Probe electrospray ionization (PESI) developed in our laboratory has been found to be tolerant to the presence of salts and buffers in sample solutions. In this report, it was examined whether PESI is applicable to the sample solutions that contain high-concentration of detergents. It was found that PESI is highly tolerant to the presence of sodium dodecyl sulphate, cetyl trimethylamminium bromide, Triton X100 and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate compared with conventional ESI and nanoESI. Therefore, PESI can be a potential analytical tool for direct analysis of protein extracts and digests containing high-concentration detergents.

Super-atmospheric pressure electrospray ion source: applied to aqueous solution.

This is a follow-up paper of our previous report on an ion source, which was operated at an operating pressure higher than the atmospheric pressure. Besides having more working gas for desolvation, the reduction of mean free path of electrons in a higher pressure environment increases the threshold voltage for gaseous breakdown, thus enabling a stable electrospray for the sample solution with high surface tension without the occurrence of electric discharge. In our previous work, the ion source was not coupled directly to the mass spectrometer and significant amount of ions were lost before entering the vacuum of the mass spectrometer. In this paper, we report the new design of our second prototype in which, by using a modified ion transport capillary, the pressurized ESI ion source was coupled directly to the first pumping stage of the mass spectrometer without additional modification on the vacuum pumping system. Demonstrations of the new ion source on the sensitive detection of native proteins from aqueous solution in both positive and negative ion modes are presented.

Sequential and exhaustive ionization of analytes with different surface activity by probe electrospray ionization.

The probe electrospray ionization (PESI) is an ESI-based ionization technique that generates electrospray from the tip of a solid metal needle. In this work, mass spectra for the single-shot PESI were measured as a function of time for a mixture of several analytes with different surface activity values. It was found that the analytes were elecrosprayed in the order of their surface activity. For example, detergent and protein were detected separately and respectively at the first and last stages of electrospray, for a mixed sample of 10(-3) M Triton X100 and 10(-5) M cytochrome c. For human breast cancer tissue, at first proteins such as α and ß chains of hemoglobin, were observed as the dominant ions, but just before the liquid droplet on the needle was depleted only lipids were observed, meaning that PESI has the advantage of the suppression effect with analytes being detected separately in the order of their surface activity values.