Coupling Electrochemistry with Probe Electrospray Ionization Mass Spectrometry

A new coupling of electrochemistry with mass spectrometry (MS) using probe electrospray ionization (PESI) is presented. Due to the high salt tolerance of PESI, the detection of electrochemical reaction products in room‐temperature ionic liquids (RTILs) is realized for the first time. Furthermore, PESI‐MS allows the analysis of electrochemical reaction products on different or multiple electrode surfaces. In addition, peptides and proteins fractionated through isoelectric focusing (IEF) in the presence of an external electric field can also be directly analyzed by using PESI‐MS, suggesting a new and rapid characterization means for the IEF technique. This study reveals the versatility of EC/PESI‐MS, which could have an impact in electrochemistry and bioanalysis fields.     

In situ analysis of soybeans and nuts by probe electrospray ionization mass spectrometry

The probe electrospray ionization (PESI) is an ESI‐based ionization technique that generates electrospray from the tip of a solid metal needle. In the present work, we describe the PESI mass spectra obtained by in situ measurement of soybeans and several nuts (peanuts, walnuts, cashew nuts, macadamia nuts and almonds) using different solid needles as sampling probes. It was found that PESI‐MS is a valuable approach for in situ lipid analysis of these seeds. The phospholipid and triacylglycerol PESI spectra of different nuts and soybean were compared by principal component analysis (PCA). PCA shows significant differences among the data of each family of seeds. Methanolic extracts of nuts and soybean were exposed to air and sunlight for several days. PESI mass spectra were recorded before and after the treatment. Along the aging of the oil (rancidification), the formation of oxidated species with variable number of hydroperoxide groups could be observed in the PESI spectra. The relative intensity of oxidated triacylglycerols signals increased with days of exposition. Monitoring sensitivity of PESI‐MS was high. This method provides a fast, simple and sensitive technique for the analysis (detection and characterization) of lipids in seed tissue and degree of oxidation of the oil samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Ambient imaging mass spectrometry by electrospray ionization using solid needle as sampling probe

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70–90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re‐dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 µm lateral resolution to form mass spectrometric images. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

The Three‐Component Biginelli Reaction: A Combined Experimental and Theoretical Mechanistic Investigation

Biginelli reactions have been monitored by direct infusion electrospray ionization mass spectrometry (ESI‐MS) and key cationic intermediates involved in this three‐component reaction have been intercepted and further characterized by tandem MS experiments (ESI‐MS/MS). Density functional theory calculations were also used to investigate the feasibility of the major competing mechanisms proposed for the Biginelli reaction. The experimental and theoretical results were found to corroborate the iminium mechanism proposed by Folkers and Johnson, whereas no intermediates directly associated with either the more energy demanding Knoevenagel or enamine mechanisms could be intercepted.     

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.     

Direct profiling of phytochemicals in tulip tissues and in vivo monitoring of the change of carbohydrate content in tulip bulbs by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which generates electrospray from the tip of a solid needle. In this study, we have applied PESI interfaced with a time of flight mass spectrometer (TOF-MS) for direct profiling of phytochemicals in a section of a tulip bulb in different regions, including basal plate, outer and inner rims of scale, flower bud and foliage leaves. Different parts of tulip petals and leaves have also been investigated. Carbohydrates, amino acids and other phytochemicals were detected. A series of in vivo PESI-MS experiments were carried out on the second outermost scales of four living tulip bulbs to monitoring the change of carbohydrate content during the first week of initial growth. The breakdown of carbohydrates was observed which was in accordance with previous reports achieved by other techniques. This study has indicated that PESI-MS can be used for rapid and direct analysis of phytochemicals in living biological systems with advantages of low sample consumption and little sample preparation. Therefore, PESI-MS can be a new choice for direct analysis/profiling of bioactive compounds or monitoring metabolic changes in living biological systems.     

Real time monitoring of accelerated chemical reactions by ultrasonication‐assisted spray ionization mass spectrometry

Ultrasonication has been used to accelerate chemical reactions. It would be ideal if ultrasonication‐assisted chemical reactions could be monitored by suitable detection tools such as mass spectrometry in real time. It would be helpful to clarify reaction intermediates/products and to have a better understanding of reaction mechanism. In this work, we developed a system for ultrasonication‐assisted spray ionization mass spectrometry (UASI–MS) with an ~1.7 MHz ultrasonic transducer to monitor chemical reactions in real time. We demonstrated that simply depositing a sample solution on the MHz‐based ultrasonic transducer, which was placed in front of the orifice of a mass spectrometer, the analyte signals can be readily detected by the mass spectrometer. Singly and multiply charged ions from small and large molecules, respectively, can be observed in the UASI mass spectra. Furthermore, the ultrasonic transducer used in the UASI setup accelerates the chemical reactions while being monitored via UASI–MS. The feasibility of using this approach for real‐time acceleration/monitoring of chemical reactions was demonstrated. The reactions of Girard T reagent and hydroxylamine with steroids were used as the model reactions. Upon the deposition of reactant solutions on the ultrasonic transducer, the intermediate/product ions are readily generated and instantaneously monitored using MS within 1 s. Additionally, we also showed the possibility of using this reactive UASI–MS approach to assist the confirmation of trace steroids from complex urine samples by monitoring the generation of the product ions. Copyright © 2014 John Wiley & Sons, Ltd.     

Dinuclear Copper Intermediates in Copper(I)‐Catalyzed Azide–Alkyne Cycloaddition Directly Observed by Electrospray Ionization Mass Spectrometry

The mechanism of the CuAAC reaction has been investigated by electrospray ionization mass spectrometry (ESI‐MS) using a combination of the neutral reactant approach and the ion‐tagging strategy. Under these conditions, for the first time, putative dinuclear copper intermediates were fished out and characterized by ESI(+)‐MS/MS. New insight into the CuAAC reaction mechanisms is provided and a catalytic cycle is proposed.     

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. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

Dimerization of ionized 4‐(methyl mercapto)‐phenol during ESI,APCI and APPI mass spectrometry

A novel ion/molecule reaction was observed to occur under electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI) conditions, leading to dimerization of ionized 4‐(methyl mercapto)‐phenol followed by fast H^· loss. The reaction is particularly favored during ESI, which suggests that this ion/molecule reaction can occur both in the solution inside the ESI‐charged droplets and in the gas‐phase environment of most other atmospheric pressure ionization techniques. The dimerization reaction is inherent to the electrolytic process during ESI, whereas it is more by ion/molecule chemistry in nature during APCI and APPI. From the tandem mass spectrometry (MS/MS) data, accurate mass measurements, hydrogen/deuterium (H/D) exchange experiments and density functional theory (DFT) calculations, two methyl sulfonium ions appear to be the most likely products of this electrophilic aromatic substitution reaction. The possible occurrence of this unexpected reaction complicates mass spectral data interpretation and can be misleading in terms of structural assignment as reported herein for 4‐(methyl mercapto)‐phenol. Copyright © 2009 John Wiley & Sons, Ltd.     

Super‐atmospheric pressure ionization mass spectrometry and its application to ultrafast online protein digestion analysis

Ion source pressure plays a significant role in the process of ionization and the subsequent ion transmission inside a mass spectrometer. Pressurizing the ion source to a gas pressure greater than atmospheric pressure is a relatively new approach that aims to further improve the performance of atmospheric pressure ionization sources. For example, under a super‐atmospheric pressure environment, a stable electrospray can be sustained for liquid with high surface tension such as pure water, because of the suppression of electric discharge. Even for nano‐electrospray ionization (nano‐ESI), which is known to work with aqueous solution, its stability and sensitivity can also be enhanced, particularly in the negative mode when the ion source is pressurized. A brief review on the development of super‐atmospheric pressure ion sources, including high‐pressure electrospray, field desorption and superheated ESI, and the strategies to interface these ion sources to a mass spectrometer will be given. Using a recent ESI prototype with an operating temperature at 220 °C under 27 atm, we also demonstrate that it is possible to achieve an online Asp‐specific protein digestion analysis in which the whole processes of digestion, ionization and MS acquisition could be completed on the order of a few seconds. This method is fast, and the reaction can even be monitored on a near‐real‐time basis. Copyright © 2016 John Wiley & Sons, Ltd.     

Elusive Reaction Intermediates in Solution Explored by ESI‐MS: Reverse Periscope for Mechanistic Investigations

Just as periscopes allow a submarine to visually search for objects above the surface of the sea, in a reversed periscope fashion electrospray mass spectrometry (ESI‐MS) can analyze the compounds at the gas phase/liquid phase interface for chemical entities which may exist in solution. The challenge is the identification and structural characterization of key elusive reaction intermediates in chemical transformations, intermediates which are able to explain how chemical processes occur. This Minireview summarizes recent selected publications on the use of ESI‐MS techniques for studying solution intermediates of homogeneous chemical reactions.     

The mechanism of the Stille reaction investigated by electrospray ionization mass spectrometry

On-line monitoring of Stille reactions was performed via direct infusion electrospray ionization mass spectrometry (ESI-MS) and its tandem version (ESI-MS/MS). When operated in the positive ion mode, ESI(+)-MS was able to transfer, directly from solution to the gas phase, the species involved in all main steps of a Stille reaction, that is, the catalytically active palladium species Pd(PPh3)2, in its molecular ion form as well as the key cationic Pd(II) intermediates, including cyclic IPd-(CH2CH)Sn species. When searching for anionic species, ESI(-)-MS monitoring showed I- as the only anion detectable in the reaction medium. A detailed catalytic cycle for a Stille reaction was elaborated in which reaction intermediates and the previously elusive catalytically active Pd(0) species are shown in association with the respective ionic species intercepted by ESI-MS and further characterized by ESI-MS/MS.     

Electrospray ionization mass spectrometry from discrete nanoliter‐sized sample volumes

We describe a method for nanoelectrospray ionization mass spectrometry (nESI‐MS) of very small sample volumes. Nanoliter‐sized sample droplets were taken up by suction into a nanoelectrospray needle from a silicon microchip prior to ESI. To avoid a rapid evaporation of the small sample volumes, all manipulation steps were performed under a cover of fluorocarbon liquid. Sample volumes down to 1.5 nL were successfully analyzed, and an absolute limit of detection of 105 attomole of insulin (chain B, oxidized) was obtained. The open access to the sample droplets on the silicon chip provides the possibility to add reagents to the sample droplets and perform chemical reactions under an extended period of time. This was demonstrated in an example where we performed a tryptic digestion of cytochrome C in a nanoliter‐sized sample volume for 2.5 h, followed by monitoring the outcome of the reaction with nESI‐MS. The technology was also utilized for tandem mass spectrometry (MS/MS) sequencing analysis of a 2 nL solution of angiotensin I. Copyright © 2010 John Wiley & Sons, Ltd.     

An overview of the retro-Diels-Alder reaction in semiunsaturated heterocyclic rings: mass spectra of new substituted 1,4,5,6,7,8-hexahydroquinolines and their oxo-analogues 5,6,7,8-tetrahydro-4H-chromenes

Electron impact ionization (EI), chemical ionization (CI), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to investigate a number of relatively large and structurally related new heterocycles such as substituted 1,4,5,6,7,8-hexahydroquinolines and their oxa-analogues 5,6,7,8-tetrahydro-4H-chromenes. In the EI spectra the hexahydroquinolines undergo the loss of the substituent attached at the C4 position, while the 4H-chromenes undergo a retro-Diels-Alder reaction (RDA) after elimination of the C4 substituent. Under chemical ionization conditions the RDA reaction is observed only for the 4H-chromenes. The ESI-MS/MS spectra reveal results similar to the EI and CI spectra, since the 4H-chromenes undergo RDA reactions while the hexahydroquinolines form a very stable even-electron pyridium ion derived from the loss of the C4 substituent.     

Electrostatic field–induced tip‐electrospray ionization mass spectrometry for direct analysis of raw food materials

Rapid characterization of metabolites and risk compounds such as chemical residues and natural toxins in raw food materials such as vegetables, meats, and edible living plants and animals plays an important part in ensuing food quality and safety. To rapidly characterize the analytes in raw food materials, it is essential to develop in situ method for directly analyzing raw food materials. In this work, raw food materials including biological tissues and living samples were placed between an electrode and mass spectrometric (MS) inlet under a strong electrostatic field; analytes were rapidly induced to generate electrospray ionization (ESI) from the sample tip by adding a drop of solvent onto the sample. Therefore, the electrostatic field–induced tip‐ESI‐MS allows raw samples to avoid contacting high voltage, and thus this method has the advantage for in vivo analysis of food living plants and animals. Metabolite profiling, residues of pesticides and veterinary drugs, and natural toxins from raw food materials have been successfully detected. The analytical performances, including the linear ranges, sensitivity, and reproducibility, were investigated for direct sample analysis. The ionization mechanism of electrostatic field–induced tip‐ESI was also discussed in this work.     

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC(50) curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were <7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.