Coupled With Electrospray Ionization Mass Spectrometry Research Articles

Nanokit coupled electrospray ionization mass spectrometry for analysis of angiotensin converting enzyme 2 activity in single living cell

Angiotensin-converting enzyme 2 (ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry (nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living hACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1–7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.

Bromine speciation in volcanic plumes: new in situ derivatization LC-MS method for the determination of gaseous hydrogen bromide by gas diffusion denuder sampling

Abstract. The chemical characterization of volcanic gas emissions gives insights into the interior of volcanoes. Bromine species have been correlated with changes in the activity of a volcano. In order to exploit the volcanic bromine gases, we need to understand what happens to them after they are outgassed into the atmosphere. This study aims to shed light on the conversion of bromospecies after degassing. The method presented here allows for the specific analysis of gaseous hydrogen bromide (HBr) in volcanic environments. HBr is immobilized by reaction with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline (EP), which acts as an inner coating inside of diffusion denuder tubes (in situ derivatization). The derivative is analyzed by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS). The collection efficiency for HBr (99.5 %), the collection efficiency for HBr alongside HCl (98.1 %), and the relative standard deviation of comparable samples (8 %) have been investigated. The comparison of the new denuder-based method and Raschig tubes as alkaline traps resulted on average in a relative bias between both methods of 10 ± 6 %. The denuder sampling setup was applied in the plume of Masaya (Nicaragua) in 2016. HBr concentrations in the range between 0.44 and 1.97 ppb were measured with limits of detection and quantification below 0.1 and 0.3 ppb respectively. The relative contribution of HBr as a fraction of total bromine decreased from 75 ± 11 % at the Santiago crater (214 m distance to the volcanic emission source) to 36 ± 8 % on the Nindiri rim (740 m distance). A comparison between our data and the previously calculated HBr, based on the CAABA/MECCA box model, showed a slightly higher trend for the HBr fraction on average than expected from the model. Data gained from this new method can further refine model runs in the future.

Probing the conformational landscape and thermochemistry of DNA dinucleotide anions via helium nanodroplet infrared action spectroscopy.

Isolation of biomolecules in vacuum facilitates characterization of the intramolecular interactions that determine three-dimensional structure, but experimental quantification of conformer thermochemistry remains challenging. Infrared spectroscopy of molecules trapped in helium nanodroplets is a promising methodology for the measurement of thermochemical parameters. When molecules are captured in a helium nanodroplet, the rate of cooling to an equilibrium temperature of ca. 0.4 K is generally faster than the rate of isomerization, resulting in "shock-freezing" that kinetically traps molecules in local conformational minima. This unique property enables the study of temperature-dependent conformational equilibria via infrared spectroscopy at 0.4 K, thereby avoiding the deleterious effects of spectral broadening at higher temperatures. Herein, we demonstrate the first application of this approach to ionic species by coupling electrospray ionization mass spectrometry (ESI-MS) with helium nanodroplet infrared action spectroscopy to probe the structure and thermochemistry of deprotonated DNA dinucleotides. Dinucleotide anions were generated by ESI, confined in an ion trap at temperatures between 90 and 350 K, and entrained in traversing helium nanodroplets. The infrared action spectra of the entrained ions show a strong dependence on pre-pickup ion temperature, consistent with the preservation of conformer population upon cooling to 0.4 K. Non-negative matrix factorization was utilized to identify component conformer infrared spectra and determine temperature-dependent conformer populations. Relative enthalpies and entropies of conformers were subsequently obtained from a van't Hoff analysis. IR spectra and conformer thermochemistry are compared to results from ion mobility spectrometry (IMS) and electronic structure methods. The implementation of ESI-MS as a source of dopant molecules expands the diversity of molecules accessible for thermochemical measurements, enabling the study of larger, non-volatile species.

Characterization of New Cyclic D,L-α-Alternate Amino Acid Peptides by Capillary Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry.

The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, impacting on their properties, which makes the choice of their monomers and their characterization a high challenge. For this purpose, we developed for the first time a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) methodology and characterized a set of eight original CP sequences of 8, 10, and 12 D,L-α-alternate amino acids with a controlled internal diameter (from 7 to 13Å) and various properties (diameter, global surface charge, hydrophobicity). This new CE-ESI-MS methodology allows verifying the structure, the purity, as well as the stability (when stored during several months) of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or pseudo sieving tools for separation purposes.

Relative ion intensities of maltooligosaccharide ethers in electrospray ionization ion trap mass spectrometry: A quantitative evaluation

Electrospray ionization coupled with mass spectrometry (ESI-MS) is an important analytical technique for the analysis of polysaccharide derivatives. It can be used to analyze substitution patterns in mixtures of oligomers to recognize heterogeneities, bimodality or blocky sequences. Quantitative analysis is hampered, however, by the fact that compounds of different chemistry and molar mass usually show different ionization efficiencies. In order to study the parameters of influence and to understand whether relative ion intensities in MS follow certain patterns. We analyzed fully O-methylated and fully O-perdeuteriomethylated oligosaccharides as two extreme cases of possible mixed O-Me-/O-Me-d3 by ESI-MS under defined conditions. Binary and complex mixtures of maltooligosaccharide ethers from DP 1 to 7 by were prepared from partially hydrolyzed per-O-methyl-β-cyclodextrin (Me-β-CD) and per-O-deuteromethyl-β-cyclodextrin (Me-d3-β-CD). Reference data for their real compositions were obtained after reductive amination by HPLC–UV. Within the applicable concentration range and with properly adjusted instrumental parameters, relative sensitivities (Me-d3/Me) in ESI-MS remained constant for a respective DP. With increasing DP, the relative sensitivity coefficients decreased from 1.01 for DP 2 to 0.78 for DP 6 for the binary mixtures and showed similar behavior for complex mixtures. The decrease in relative sensitivity coefficients for corresponding Me-d3 and Me derivatives has been overcome by m-amino-benzoic acid labeling and ESI-MS in negative mode or by measuring the original mixtures in nano-ESI-MS. Thus, discrimination probably caused by differences in surface activity and electrophoretic mobility can be leveled by an appropriate tag or formation of smaller droplets as in nano-ESI.

Organosulfates in cloud water above the Ozarks' isoprene source region

Secondary organic aerosol formation via aqueous processing, particularly from the oxidation of biogenic volatile organic compounds, is hypothesized to contribute significantly to the global aerosol burden. In this study, electrospray ionization coupled with mass spectrometry (ESI-MS) was utilized to detect organosulfates and oligomers in cloud water collected in July above the Missouri Ozarks, an environment significantly influenced by isoprene oxidation. Community Multiscale Air Quality (CMAQ) modeling suggested that the aerosol at cloud height was characterized by high water, sulfate, and biogenic secondary organic aerosol content, conducive to aqueous-phase processing and organosulfate formation. CMAQ modeling also suggested the presence of gas-phase organic peroxides and nitrates, which can partition into the particle-phase and form organosulfates. Several potential organosulfates from isoprene, monoterpene, and sesquiterpene oxidation were detected in the cloud water. In particular, the ubiquitous organosulfate C5H12O7S (detected by ESI-MS at m/z −215), derived from isoprene epoxydiols, was detected. These results highlight the role of aqueous-phase reactions in biogenic SOA formation and cloud processes in isoprene oxidation-influenced regions.

Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpioL.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study.

Potential Diagnostic Assay for Cystinuria by Capillary Electrophoresis Coupled to Mass Spectrometry

Cystinuria is an autosomal recessive genetic disorder characterized by abnormal intestinal and renal tubular transport of L-cystine as well as of L-lysine, L-arginine and L-ornithine. This leads to excessive urinary excretion of amino acids, with the formation of kidney stones caused by the low solubility of L-cystine in the urine. In this study, an analytical method for simultaneous determination of these four amino acids in urine by capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) was developed and validated. Using standard solutions of L-cystine, L-lysine, L-arginine and L-ornithine, the amino acid detection limits by this method were 114.2, 61.3, 72.7 and 86.7 µmol L-1. Standard solutions were injected in a silica capillary column (50 µm i.d. and 70 cm length) under 2 psi of pressure by 10 s. The separation occurred at 300 V cm-1, using 1.0 mol L-1 formic acid in 10% methanol in water as the background electrolyte. The method was applied to the urine of a patient clinically diagnosed as a cystinuria carrier, which revealed the presence of 900.5 ± 5, 600.0 ± 2, 700.2 ± 1 and 500.0 ± 3 µmol L-1 of amino acid, respectively, and 75.3 ± 1 µmol L-1 of creatinine. The CE-ESI-MS method described here for analyzing L-cystine and other cystinuria-related amino acids is a sensitive and reliable diagnostic tool for characterizing and monitoring this disease.

QuEChERS method for the extraction of isoflavones from soy-based foods before determination by capillary electrophoresis-electrospray ionization-mass spectrometry

This article reports the development of a modified approach of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology for the extraction of naturally occurring substances from solid soy-based products. The use of this methodology offers advantages such as simplicity and ease of use, especially taking into account the complexity of food matrices. Here, capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) was applied for the separation and quantification of isoflavones in soy products. Because of the importance of the different forms of isoflavones as regards their biological activity, a method that would permit both aglycones and the glycoside forms of isoflavones to be determined was developed. The proposed method was successfully applied to the analysis of the glycosylated forms glycitin, daidzin and genistin, and the aglycones formononetin, biochanin A, glycitein, daidzein, and genistein in commercially available soy biscuit samples. The parameters evaluated for the validation of the method developed included the linearity of the calibration plots, the detection and quantification limits, the existence of matrix effect(s), repeatability, reproducibility and recovery. The results of the validation process revealed that the proposed method is a possible alternative in routine analysis of isoflavones in food matrices.

The structural diversity of lipopolysaccharide expressed by non-typeable Haemophilus influenzae strains 1158 and 1159

A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSn on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-d-α-d-Hepp-(1→6)-β-d-Glcp linked to HepI from the common structural element of H. influenzae LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of β-d-Glcp attached to HepIII and was also located at a novel position linked to d-α-d-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MSn experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the d,d-Hep to the outer core region in strain 1158.

Chip-Type Asymmetrical Flow Field-Flow Fractionation Channel Coupled with Mass Spectrometry for Top-Down Protein Identification

A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.

Programmed Nebulizing Gas Pressure for Efficient and Stable Capillary Electrophoresis−Mass Spectrometry Analysis of Anionic Compounds in Positive Separation Mode

Here we have developed a method for the analysis of anionic compounds by capillary electrophoresis coupled to electrospray ionization-mass spectrometry (CE-ESI-MS). The strategy proposed is based on the application of programmed nebulizing gas pressure (PNP), which is applied during the different steps of analysis: sample injection, electrophoretic separation, and detection by the mass spectrometer. The proposed procedure prevents the frequent drops in current observed in the analysis of anions and allows high separation efficiency by capillary electrophoresis to be maintained because it is not necessary to employ pressured-assisted electrophoresis. Additionally, for the first time we describe the use of 100 μm capillaries in capillary electrophoresis-mass spectrometry, allowing the loading of larger samples and hence greater sensitivity, with no loss of the efficiency achieved with the 50 μm capillaries usually used.

On‐line capillary electrophoresis‐electrospray ionization mass spectrometry analysis of urinary porphyrins

A rapid, high-resolution and high-efficiency novel method for the simultaneous determination of mesoporphyrin (MP), coproporphyrin (CP) and uroporphyrin (UP) in human urine by capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) was developed. The simple laboratory-made interface was developed by using a micro-tee connection and a liquid junction. The porphyrins were well separated by CE with a positive 12 kV voltage in a 20 mM ammonium acetate buffer (pH 10.5) containing 40% v/v acetonitrile. The sheath liquid was composed of acetonitrile-water (50:50 v/v) containing 5 mM ammonium acetate and 0.1% v/v formic acid (pH 3.0) with a flow rate of 5 microL/min. The porphyrins were identified with the criteria of migration time and m/z values, and quantified with the measurement of peak area of [M+H]+ ion. Limits of detection for MP, CP and UP were 85 nM, 140 nM and 300 nM, respectively. The correlation coefficients of calibration curves were between 0.9956 and 0.9862. The intra-day precisions in terms of relative standard deviations (n = 12) of the method were 1.1-3.9% for migration times and 2.3-14.1% for peak areas. The porphyrin recoveries of spiked urine samples were between 73% and 96%. The liquid junction interface for CE-ESI-MS was demonstrated to be applicable for the potential quantitative analysis of clinical samples.

Simultaneous determination of new thioamphetamine designer drugs in plasma by capillary electrophoresis coupled with mass spectrometry

A simple method for the simultaneous identification and quantification of four 2,5-methylenedioxy derivatives of 4-thioamphetamine (ALEPH series) in plasma samples was developed. The method consists of solid-phase extraction (SPE) using a Bond Elut C(18) cartridge and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE/ESI-MS). The SPE method used required only simple steps and provided a clean extract from which identification of each drug was feasible, even at low concentrations. The method was validated according to international guidelines. The calibration curves were linear over the concentration range of 50 to 1000 ng/mL for all drugs with correlation coefficients that exceeded 0.998. The lower limits of detection of the drugs were 23-43 ng/mL. The absolute recoveries for the drugs were 64-92% and 75-96% at concentrations of 100 and 500 ng/mL, respectively. The validation data (precision, accuracy, and recovery) show the reproducibility and selectivity of the method. This clean and simple method allows the routine detection of designer drugs such as thioamphetamines which may become a serious problem in the control of illegal drugs.

Determination of 4-alkyl 2,5 dimethoxy-amphetamine derivatives by capillary electrophoresis with mass spectrometry detection from urine samples

The methylenedioxy-derivatives of amphetamine represent the largest group of designer drugs. The 4-methyl (DOM), -ethyl (DOET) and -propyl (DOPR) derivatives of 2,5-dimethoxy-amphetamine (2,5-DMA) were found to possess quite similar serotonin receptor affinities [R.A. Glennon, D.L. Doot, R. Young, Pharmacol. Biochem. Behav. 14 (1981) 287.]. This paper describes a method to screen for and quantify DOM, DOET and DOPR in urine samples, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validated according to international guidelines. Data for accuracy and precision were within required limits. Calibration curves were generated ranging from 10 to 1000 ng/mL and correlation coefficients always exceeded 0.996.

Determination of four thiophenethylamine designer drugs (2C-T-series) in human plasma by capillary electrophoresis with mass spectrometry detection

In recent years, the frequent appearance of phenethylamine designer drugs on the illicit drug market has been a matter of concern for all authorities involved. New phenethylamine drugs are being introduced because these compounds are not covered by existing legislation. Therefore, the new drugs cannot be considered illicit drugs until their names are officially recognized. This paper describes a method to screen for and quantify four 2,5-methylenedioxy-derivatives of 4-thio-phenethylamine (2C-T-series) in human plasma, using capillary electrophoresis coupled with electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple liquid extraction was used for sample cleanup. The method was validated according to international guidelines.

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry.

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.