Ultrasonication-assisted spray ionization mass spectrometry for the analysis of biomolecules in solution

In this paper, we describe a novel technique—ultrasonication-assisted spray ionization (UASI)—for the generation of singly charged and multiply charged gas-phase ions of biomolecules (e.g., amino acids, peptides, and proteins) from solution; this method employs a low-frequency ultrasonicator (ca. 40 kHz) in place of the high electric field required for electrospray ionization. When a capillary inlet is immersed into a sample solution within a vial subjected to ultrasonication, the solution is continually directed to the capillary outlet as a result of ultrasonication-assisted capillary action; an ultrasonic spray of the sample solution is emitted at the outlet of the tapered capillary, leading to the ready generation of gas-phase ions. Using an ion trap mass spectrometer, we found that singly charged amino acid and multiply charged peptides/proteins ions were generated through this single-step operation, which is both straightforward and extremely simple to perform. The setup is uncomplicated: only a low-frequency ultrasonicator and a tapered capillary are required to perform UASI. The mass spectra of the multiply charged peptides and proteins obtained from sample solutions subjected to UASI resemble those observed in ESI mass spectra.     

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.     

Ultrasonication-assisted spray ionization mass spectrometry for on-line monitoring of organic reactions

A straightforward on-line monitoring of organic reactions by ultrasonication-assisted spray ionization mass spectrometry (UASI MS) is demonstrated in this work.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Semi‐online nanoflow liquid chromatography/matrix‐assisted laser desorption ionization mass spectrometry of synthetic polymers using an octadecylsilyl‐modified monolithic silica capillary column

We have designed a semi‐online liquid chromatography/matrix‐assisted laser desorption/ionization mass spectrometry (LC/MALDI‐MS) system to introduce eluent from a octadecylsilyl (ODS) group modified monolithic silica capillary chromatographic column directly onto a sample plate for MALDI‐MS analysis. Our novel semi‐online system is useful for rapidly and sensitively examining the performance of a monolithic capillary column. An additional advantage is the small elution volume of a monolithic capillary column, which allows delicate eluents, such as 1,1,1,3,3,3,‐hexafluoroisopropyl alcohol (HFIP), to be used to achieve cost‐effective analysis. Using the semi‐online LC/MALDI‐MS system, chromatographic separation of polymers by the monolithic column with different eluents was studied. Separation of poly(methyl methacrylate) and Nylon 6/6 showed that the column functioned via size‐exclusion separation when tetrahydrofuran or HFIP eluent was used. On the other hand, the separation behavior of Nylon 11 indicated a reversed‐phase mode owing to the interaction of the polymer with the modified ODS group in the column. Using tetrahydrofuran/methanol (1:1, v/v) as the eluent, the LC/MALDI‐MS spectra of poly(lactic acid), which contains both linear and cyclic polymer structures, showed that the column could separate the hydrophobic cyclic polymer and elute it out relatively slowly. The monolithic column functions basically via size‐exclusion separation; the reversed‐phase separation by interaction with the ODS functions may have less influence on column separation. The semi‐online monolithic capillary LC/MALDI‐MS method we have developed should provide a means of effectively analyzing synthetic polymers. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of lipids with desorption atmospheric pressure photoionization‐mass spectrometry (DAPPI‐MS) and desorption electrospray ionization‐mass spectrometry (DESI‐MS)

In this article, the effect of spray solvent on the analysis of selected lipids including fatty acids, fat‐soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids has been studied by two different ambient mass spectrometry (MS) methods, desorption electrospray ionization‐MS (DESI‐MS) and desorption atmospheric pressure photoionization‐MS (DAPPI‐MS). The ionization of the lipids with DESI and DAPPI was strongly dependent on the spray solvent. In most cases, the lipids were detected as protonated or deprotonated molecules; however, other ions were also formed, such as adduct ions (in DESI), [M‐H]⁺ ions (in DESI and DAPPI), radical ions (in DAPPI), and abundant oxidation products (in DESI and DAPPI). DAPPI provided efficient desorption and ionization for neutral and less polar as well as for ionic lipids but caused extensive fragmentation for larger and more labile compounds because of a thermal desorption process. DESI was more suitable for the analysis of the large and labile lipids, but the ionization efficiency for less polar lipids was poor. Both methods were successfully applied to the direct analysis of lipids from pharmaceutical and food products. Although DESI and DAPPI provide efficient analysis of lipids, the multiple and largely unpredictable ionization reactions may set challenges for routine lipid analysis with these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Multi‐mycotoxin analysis in complex biological matrices using LC‐ESI/MS: Experimental study using triple stage quadrupole and LTQ‐Orbitrap

In the present study, we report the application of LC‐MS based on two different LC‐MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC‐MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC‐ESI‐MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra‐assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro‐LC‐LTQ‐Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro‐LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.     

Continuous‐flow extractive desorption electrospray ionization coupled to normal phase separations and for direct lipid analysis from cell extracts

Normal phase liquid chromatography is a common mode for chiral separations. Many chiral amines are used as drugs or are important intermediates for drug synthesis. Electrospray ionization mass spectrometry is well known for its high sensitivity. However, when using normal phase liquid chromatography, electrospray ionization is hampered by the poor ionization efficiency of analytes from organic eluents. Continuous‐flow extractive desorption electrospray ionization, which introduces the eluents through a hypodermic needle into the electrospray plume is demonstrated here for its success to interface normal phase liquid chromatography to mass spectrometry detection. Such an approach was shown to be as or more sensitive than ultraviolet detection for a selected set of aromatic amine‐functionalized enantiomers. Also demonstrated is the direct infusion of cell extracts to monitor phospholipids from three different bacterial cells. Despite their presence in non‐electrospray‐ionization‐friendly extraction solvents, continuous‐flow extractive desorption electrospray ionization enabled the sensitive detection of phospholipids and the ability to tune ion forms through incorporation of different spray modifiers.     

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.     

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI‐MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI‐MS). A cold spray ionization providing a stable solvation‐ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug‐protein complexes with exact stoichiometries. In this paper, we apply CSI‐MS to explore the interactions of ginsenosides toward amyloid‐β‐peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI‐MS and ESI‐MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI‐MS, while dehydration products are more readily available by ESI‐MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI‐MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.     

A dopant for improved sensitivity in easy ambient sonic‐spray ionization mass spectrometry

Recently, 3‐nitrobenzonitrile (3‐NBN) has been used to improve sensitivity of sonic‐spray ionization mass spectrometry. Easy ambient sonic‐spray ionization (EASI) is one of the simplest, gentlest and most used spray‐based desorption/ionization ambient techniques, but limited sensitivity has been commonly taken as its major drawback. Herein we investigate the use of 3‐NBN as a dopant in EASI‐MS for improved sensitivity. Using a few typical EASI samples as test cases, the presence of 10 ppm (µg ml^?1) of 3‐NBN in the spray solvent showed two to fourfold gains in EASI‐MS sensitivity as measured both by total ion current and S/N ratios, accompanied with significant reductions in chemical noise. Sensitivity for DESI using 3‐NBN as a dopant also improved and dopant DESI versus dopant EASI sensitivities were compared. The use of solvent dopants seems therefore to be a promising strategy to improve sensitivity for spray‐based ambient MS techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Collision‐induced dissociation study of poly(2‐ethyl‐2‐oxazoline) using survival yields and breakdown curves

Poly(2‐ethyl‐2‐oxazoline), a synthetic polymer was analysed by mass spectrometry using different ion sources. Two distributions could be identified in the mass spectra which related to two different polymer series (one with hydrogen and hydroxyl end‐groups and the other with methyl and hydroxyl end‐groups). The fragmentation behaviour of the protonated oligomers was studied in a quadrupole time‐of‐flight mass spectrometer (MS) with electrospray, atmospheric pressure chemical ionization and direct analysis in real time soft ionization techniques. Three product ion series were identified in the MS/MS spectra independently of the ion source used. Based on the results, a mechanism was proposed for the dissociation by means of the accurate mass of the product ions, pseudo MS³ experiments and the energy dependence of the product ion intensity, i.e. breakdown curves. The survival yield method was used to highlight the correlation between the size of the oligomers and the laboratory frame collision energy. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid screening and determination of 4‐chloroamphetamine in saliva by paper spray‐mass spectrometry and capillary electrophoresis‐mass spectrometry

A novel drug‐screening system, consisting of paper spray‐MS (PS‐MS) and a CE‐ESI‐MS method was developed. This system can be easily switched either to PS‐MS for rapidly screening samples or to the traditional CE‐ESI‐MS method for separation and to obtain detailed mass spectral information, while sharing the same mass spectrometer. In the former case, when a sharp (15°‐tip) chromatography paper was used, the optimized distance from the paper tip to the mass inlet was 7.7 mm, whereas the optimized distance for the CE‐ESI tip was ～13.5 mm. Using 4‐chloroamphetamine as a model compound, the LODs for PS‐MS and CE‐ESI‐MS were determined to ～0.1 and 0.25 ppm, respectively. Comparisons of results obtained using PS‐MS and CE‐ESI‐MS and the experimental conditions are described.     

On‐Demand Ambient Ionization of Picoliter Samples Using Charge Pulses

Relay electrospray ionization (rESI) from a capillary containing a sample solution (or from an array of such capillaries) is triggered by charge deposition onto the capillary. Suitable sources of primary ions, besides electrosprays, are plasma ion and piezoelectric discharge plasma sources. With no requirement for physical contact, high‐throughput sample screening is enabled by rapidly addressing individual secondary (sample) capillaries. Sub‐pL sample volumes can be loaded and sprayed. Polar analytes, including neurotransmitters, phosphopeptides, oligonucleotides, illicit drugs, and pharmaceutical compounds are successfully ionized by rESI with concentration sensitivities (0.1 ppb for acetylcholine) which are similar to nanoESI but absolute sensitivities are orders of magnitude better. Nonpolar analytes (steroids, alkynes) are ionized by rESI using an open‐tube secondary capillary and injecting electrolytically generated metal cations from the primary electrospray.     

A microfluidic reactor for rapid,low‐pressure proteolysis with on‐chip electrospray ionization

A microfluidic reactor that enables rapid digestion of proteins prior to on‐line analysis by electrospray ionization mass spectrometry (ESI‐MS) is introduced. The device incorporates a wide (1.5 cm), shallow (10 µm) reactor ‘well’ that is functionalized with pepsin‐agarose, a design that facilitates low‐pressure operation and high clogging resistance. Electrospray ionization is carried out directly from a short metal capillary integrated into the chip outlet. Fabrication, involving laser ablation of polymethyl methacrylate (PMMA), is exceedingly straightforward and inexpensive. High sequence coverage spectra of myoglobin (Mb), ubiquitin (Ub) and bovine serum albumin (BSA) digests were obtained after <4 s of residence time in the reactor. Stress testing showed little loss of performance over ～2 h continuous use at high flow rates (30 µL/min). The device provides a convenient platform for a range of applications in proteomics and structural biology, i.e. to enable high‐throughput workflows or to limit back‐exchange in spatially resolved hydrogen/deuterium exchange (HDX) experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Real‐time reaction monitoring by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI‐MS was used to monitor some biological and chemical reactions in real‐time, such as acid‐induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI‐MS, time‐resolved mass spectra and ion chromatograms can be obtained reproducibly. Real‐time PESI‐MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI‐MS can be considered as a potential tool for real‐time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd.     

Electron ionization LC‐MS with supersonic molecular beams—the new concept,benefits and applications

A new type of electron ionization LC‐MS with supersonic molecular beams (EI‐LC‐MS with SMB) is described. This system and its operational methods are based on pneumatic spray formation of the LC liquid flow in a heated spray vaporization chamber, full sample thermal vaporization and subsequent electron ionization of vibrationally cold molecules in supersonic molecular beams. The vaporized sample compounds are transferred into a supersonic nozzle via a flow restrictor capillary. Consequently, while the pneumatic spray is formed and vaporized at above atmospheric pressure the supersonic nozzle backing pressure is about 0.15 Bar for the formation of supersonic molecular beams with vibrationally cold sample molecules without cluster formation with the solvent vapor. The sample compounds are ionized in a fly‐though EI ion source as vibrationally cold molecules in the SMB, resulting in ‘Cold EI’ (EI of vibrationally cold molecules) mass spectra that exhibit the standard EI fragments combined with enhanced molecular ions. We evaluated the EI‐LC‐MS with SMB system and demonstrated its effectiveness in NIST library sample identification which is complemented with the availability of enhanced molecular ions. The EI‐LC‐MS with SMB system is characterized by linear response of five orders of magnitude and uniform compound independent response including for non‐polar compounds. This feature improves sample quantitation that can be approximated without compound specific calibration. Cold EI, like EI, is free from ion suppression and/or enhancement effects (that plague ESI and/or APCI) which facilitate faster LC separation because full separation is not essential. The absence of ion suppression effects enables the exploration of fast flow injection MS‐MS as an alternative to lengthy LC‐MS analysis. These features are demonstrated in a few examples, and the analysis of the main ingredients of Cannabis on a few Cannabis flower extracts is demonstrated. Finally, the advantages of EI‐LC‐MS with SMB are listed and discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Coupling of hydrodynamically closed large bore capillary isotachophoresis with electrospray mass spectrometry

Capillary isotachophoresis with coupled columns provides efficient means for rapid electrophoretic analysis of sample volumes of up to 10 μl or more. Commercially available instruments are commonly equipped with conductivity and UV absorbance detectors; however, their on-line coupling with electrospray mass spectrometry is highly desirable. In this work we have modified the commercial coupled column isotachophoresis system for direct connection to an ion trap mass spectrometer. The design included attachment of an elution block with a short capillary transfer line directing the separated zones towards the mass spectrometer. The modification further included separation of the injection and electrode blocks from the separation columns by semipermeable membranes eliminating unwanted fluid movements in the wide bore fluoropolymer separation capillaries. Fused silica capillaries with varying internal diameter were connected as a transfer line between the elution block and mass spectrometer. The transfer line served also as the ESI tip of the sheathless electrospray interface. During the analysis the first, wide bore preseparation capillary with 0.8 mm internal diameter served for removal of the bulk sample components and preseparation of the potentially interfering analytes. After the electronic column switching the separation was finished in a 0.3 mm internal diameter capillary and the separated ITP zones were transferred in-line by a spray liquid towards the mass spectrometer. The instrumentation was tested for determination of vitamins in whole blood analysis and separation of tryptic peptides.     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010