Electrospray Ionization Process Research Articles

Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry

Electrospray ionisation (ESI) is renowned for its ability to ionise intact proteins for sensitive detection by mass spectrometry (MS). However, the use of a conventional direct current ESI voltage can result in the formation of relatively large initial droplet sizes, which can limit efficient ion desolvation and sensitivity. Here, pulsed nanoESI (nESI) MS using nanoscale emitters with inner diameters of ~250 nm is reported. In this approach, the nESI voltage is rapidly pulsed from 0 to ~1.5 kV with sub-nanosecond rise times, duty cycles from 10 to 90%, and repetition rates of 10 to 350 kHz. Using pulsed nESI, the performance of MS for the detection of intact proteins can be improved in terms of increased ion abundances and decreased noise. The absolute ion abundances and signal-to-noise levels of protonated ubiquitin, cytochrome C, myoglobin, and carbonic anhydrase II formed from standard denaturing solutions can be increased by up to 82% and 154% using an optimal repetition rate of ~200 kHz compared to conventional nESI-MS. Applying pulsed nESI-MS to a mixture of four proteins resulted in the signal for each protein increasing by up to 184% compared to the more conventional nESI-MS. For smaller ions (≤1032 m/z), the signal can also be increased by the use of high repetition rates (200–250 kHz), which is consistent with the enhanced performance depending more on general factors associated with the ESI process (e.g., smaller initial droplet sizes and reduced Coulombic repulsion in the spray plume) rather than analyte-specific effects (e.g., electrophoretic mobility). The enhanced sensitivity of pulsed nESI is anticipated to be beneficial for many different types of tandem mass spectrometry measurements.

Understanding the electrospray ionization response factors of per- and poly-fluoroalkyl substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are used extensively in commercial products. Their unusual solubility properties make them an ideal class of compounds for various applications. However, these same properties have led to significant contamination and bioaccumulation given their persistence in the environment. Development of analytical techniques to detect and quantify these compounds must take into account the potential for these properties to perturb these measurements, specifically the potential to bias the electrospray ionization (ESI) process. Direct injection ESI analysis of 23 different PFAS species revealed that hydrophobicity and PFAS class can predict the ESI overall response factors. In this study, a method for predicting the behavior of individual PFAS compounds, including relative retention order in chromatography, is presented which is simply based on the number of fluorine atoms in the molecule as well as the class of the compound (e.g., perfluroalkylcarboxylic acids) vs. computational estimations (e.g., non-polar surface area and logP).

On-Demand Mass Spectrometry Analysis by Miniature Mass Spectrometer.

Electrospray ionization (ESI) has become a powerful tool for the analysis of biomolecules by mass spectrometry (MS). The process of ESI is difficult to control, and side reactions such as electrochemical reactions can occur during the ESI process because of the high voltages applied. Herein, a novel on-demand MS analysis method was developed based on discontinuous ion injection-induced ESI on a miniature MS system. Highly efficient ionization was enabled under low voltages (<300 V) using a discontinuous atmospheric pressure interface. On-demand ionization showed comparable sensitivity with regular nanoESI for the analyses of a series of compounds. It was found to be softer than regular ESI or nanoESI methods for ionization of proteins such as myoglobin and cytochrome C. As the ionization finished as soon as the interface was closed, the sample consumption was observed to reduce significantly for MS analysis, allowing single-cell analysis with multiple MS and MS/MS measurements.

Electrospray deposition as a smart technique for laccase immobilisation on carbon black-nanomodified screen-printed electrodes

Enzymes immobilisation represents a critical issue in the design of biosensors to achieve standardization as well as suitable analytical performances in terms of sensitivity, selectivity, and stability. In this work electrospray deposition (ESD) has been exploited as a novel technique for the immobilisation of laccase enzyme on carbon black modified screen-printed electrodes. The aim is to fabricate an amperometric biosensor for phenolic compound detection. The electrodes produced by ESD have been analysed by scanning electron microscopy and characterised electrochemically to prove that this immobilisation technique is suited to manufacture high performance biosensors. The results show that the laccase enzyme maintains its activity after undergoing the electrospray ionisation process and deposition and the fabricated biosensor has improved performances in terms of storage (up to 3 months at room temperature) and working (up to 25 measurements on the same electrode) stability. The laccase-based biosensor has been tested for phenolic compound detection, with catechol as target analyte, in the linear range 2.5–50 μM, with 2.0 μM limit of detection, without interference from lead, cadmium, atrazine, and paraoxon, and without matrix effect in drinking, surface, and wastewater.

Detection of ergosterol using liquid chromatography/electrospray ionization mass spectrometry: Investigation of unusual in-source reactions.

In the field of natural products, de-replication of complex mixtures has become a usual practice to annotate known compounds and avoid their re-isolation. For this purpose, many groups rely on liquid chromatography coupled to high-resolution mass spectrometry (HPLC/MS) to deduce molecular formulae of compounds allowing comparison with public or in-house databases. Electrospray ionization (ESI) is usually considered as the method of choice for investigating a large panel of compounds but, in some cases, it may lead to unusual results as described in this article for ergosterol. Ergosterol and other fungal sterols in methanolic solution were analysed using various chromatographic gradients with HPLC/MS using both ion trap time-of-flight MS and Orbitrap MS instruments fitted with an ESI source. Further flow injection analyses were performed to investigate the influence of the solvent composition. MS/MS fragmentation data were acquired to annotate the various ions observed. Contrary to other fungal sterols, ergosterol was found to be highly sensitive to oxidation during ESI. Putative structures were proposed based on MS/MS studies and known oxidation mechanisms of ergosterol by reactive oxygen species that could be formed in the ESI process. The proportion of acetonitrile in the eluent was found to influence this in-source oxidation, with an increased proportion of oxidized sodium adducts with higher proportions of acetonitrile. While ergosterol is a major sterol found in fungi, this study investigates its ionization by electrospray for the first time. The results reported here will help further detection and annotation of this compound in fungal extracts after HPLC/ESI-MS analyses.

Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers.

Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.

The fallacy of using solvent extraction for the study of metal/ligand complexation in mixed solvent systems

Metal complexes can be identified by ion mobility mass spectrometry, often using mixed aqueous solvents, which improve the electrospray ionization process but induce significant changes in thermodynamic values associated with metals and ligands. This work demonstrates that the addition of 10% acetonitrile and 1.0% dimethyl sulfoxide leads to an increased autoprotolysis constant, does not influence the nitrilotriacetic acid pKa values, but decreases europium extraction in a solvent extraction system. This work shows the inability to quantify the stability constant of Eu/NTA complex in mixed solvent using a solvent extraction technique, as polar solvents are dissolved in the dodecane organic phase.

Behavior of transition metal salts during the electrospray ionization process

A study of the behavior of transition metal salts during the electrospray ionization (ESI) process using a quadrupole ion trap mass spectrometer and novel ion source is reported. The unique results in both positive and negative modes for multiple salts of 28 different cations are discussed. In positive mode, the metal cations formed clusters with the solvents, following the pattern [M + (solvent)n]+, with maximum observed values for n of 5. In general, transition metals with more than one oxidation state, such as iron, cobalt, and chromium, produced more complicated mass spectra, because the ions formed also included multiples of the anion. In negative mode, the cations formed clusters with the anions of the salts, following the pattern [My + Xn]-. The relationship between y and n correlates to the oxidation state of the cation, with maximum observed values for y and n of 3 and 7, respectively. For some environmental applications, studies in negative mode demonstrate an alternative to specialized elemental analysis instrumentation such as ICP/MS or AMS, as well as complicated sample preparation, such as the use of chelating complexes. This is especially applicable for the analyses of mercury and lead, as these species form only a few, predictable ions. For instance, mercury present in seawater forms a single ion, [Hg + Cl3]-, which can be readily isolated from the sodium chloride clusters in the background with a non-specialized ion trap mass spectrometer using ESI, and can be verified by its characteristic isotopic pattern.

Conformers of Ubiquitin 6+ for Different Charge Distributions: Atomistic Structures and Ion Mobility Cross Sections.

Despite the fact that protein chemistry occurs in solution, studying the structure of desolvated proteins in mass spectrometry vacuum conditions allows one to study biological molecules in complete isolation. The electrospray ionization process results in charged protein conformers for which the charge locations distribution and the corresponding structure are not known. In this paper, we explore the influence of different charge distributions on the structure of conformers of ubiquitin 6+ in the vacuum conditions of mass spectrometry. Using the Tinker implementation of the AMBER99FF force field, the atomistic lowest-energy structures for those charge distributions were calculated by the DEEPSAM algorithm. Differences among the calculated structures are analyzed and discussed in terms of the relation to experimentally observable ion mobility mass spectrometry cross sections. They also are compared to the neutral ubiquitin's native structure and to the nearly linear structure of the ubiquitin 13+ ion. In general, different charge distributions lead to substantially different structures and ion mobility cross sections. In this study, however, it was found that this is not always the case. Our conclusion is that measurements of ion mobility cross sections cannot be used to help determine a unique charge distribution because different charge distributions lead to structures that approximately agree in terms of experimental cross sections.

Assessing mixtures of supercharging agents to increase the abundance of a specific charge state of Neuromedin U

With increasing evidence of the important role of peptides in pathophysiological processes, a trend towards the development of highly sensitive bioanalytical methods is ongoing. Inherent to the electrospray ionization process of peptides and proteins is the production of multiple charge states which may hamper proper and sensitive method development. Supercharging agents allow modifying the maximal charge state and the corresponding distribution of charges, thereby potentially increasing the number of ions reaching the detector in selected reaction monitoring mode. In this study, the use of mixtures of charge state modifying additives, i.e. m-nitrobenzylalcohol (mNBA), sulfolane and dimethyl sulfoxide (DMSO), to specifically increase the abundance of one charge state of interest has been investigated. Screening experiments were performed to define an experimental domain, which was then further investigated via a response surface design to predict the optimal combination and concentration of superchargers. Using a combination of mNBA and DMSO (0.008% and 0.5% m/v respectively), we were able to increase the abundance of the +4 charge state of the investigated peptide neuromedin U from 64% to 87%. Unfortunately, charge state coalescence did not result in repeatable sensitivity improvements in this case study. However, it remains an attractive approach during method development of peptide bioanalytical methods, as coalescence to a particular intermediate charge state is difficult to obtain by using only one supercharging agent.

Electrospray Ionization of Polypropylene Glycol: Rayleigh-Charged Droplets, Competing Pathways, and Charge State-Dependent Conformations.

Recent molecular dynamics (MD) simulations from various laboratories have advanced the general understanding of electrospray ionization (ESI)-related processes. Unfortunately, computational cost has limited most of those previous endeavors to ESI droplets with radii of ∼3 nm or less, which represent the low end of the size distribution in the ESI plume. The current work extends this range by conducting simulations on aqueous ESI droplets with radii of 5.5 nm (∼23 000 water molecules). Considering that computational cost increases with r6, this is a significant step forward. We focused on the ESI process for polypropylene glycol (PPG) which is a common ESI-MS calibrant. Different chain lengths (PPG10, 30, and 60) were tested in droplets that were charged with excess Na+. Solvent evaporation and Na+ ejection, with occasional progeny droplet formation, kept the systems at 80-100% of the Rayleigh limit throughout their life cycle. PPG chains migrated to the droplet surface where they captured Na+ via binding to ether oxygens. Various possible pathways for PPG release into the gas phase were encountered. Some PPG10 runs showed ejection from the droplet surface, consistent with the ion evaporation model (IEM). In other instances, PPG was released after near-complete solvent evaporation, as envisioned by the charged residue model (CRM). A third avenue was the partial separation from the droplet to form double or single-tailed structures, with subsequent chain detachment from the droplet. This last pathway is consistent with the chain ejection model (CEM). Immediately after detachment many chains were electrostatically stretched, but they subsequently collapsed into compact conformers. Extended structures were retained only for the most highly charged ions. Our simulations were complemented by ESI-MS and ion mobility measurements. MD-predicted charge states and collision cross sections were in agreement with these experimental data, supporting the mechanistic insights obtained.

Two-dimensional capillary electrophoresis-mass spectrometry (CE-CE-MS): coupling MS-interfering capillary electromigration methods with mass spectrometry.

Electromigration separation techniques often demand certain compounds in the electrolyte to achieve the required selectivity and efficiency. These compounds, including the electrolyte itself, ampholytes, polymeric compounds for sieving, complexing agents, tensides, etc. are often non-volatile. Thus, interference with the electrospray ionization process is a common issue, impeding direct coupling of such electrolyte systems to mass spectrometry. Still, several options exist to obtain mass spectra after separation, including offline fractionation, alternative ionization, dilution, or the change to volatile constituents. In the first part of this article, these methods are discussed. However, all of these options are a compromise of separation performance and sensitivity of mass spectrometric detection. Two-dimensional capillary electrophoresis-mass spectrometry (CE-CE-MS) systems represent a promising alternative to the aforementioned challenges, as they allow the use of existing methods with best separation performance in combination with sensitive mass characterization. In this context, the second part of this article is dedicated to the advantages, limitations, and applications of this approach. Finally, an outlook towards future developments is given.

Micelles Protect Intact Metallo-supramolecular Block Copolymer Complexes from Solution to Gas Phase during Electrospray Ionization

Supramolecular diblock copolymers using metal-ligand coordination can be synthesized under ambient conditions by delicate design of the end groups of the homopolymer chains. However, mass spectrometric analysis of such metallo-supramolecular copolymers is challenging. One of the reasons is the nonpolarity of the polymer chains, making it hard to disperse the complexes in electrospray ionization (ESI)-friendly environments. The other difficulty is the noncovalent nature of such copolymers, which is easily disrupted during the ionization. Here, we demonstrate that the intact metallo-supramolecular diblock copolymers can be maintained sufficiently during the ESI process in aqueous solution within micelles. The high-resolution mass spectrometric evidence revealed that the surfactant molecules effectively protect the noncovalent binding of the complexes into gaseous ions. Intriguingly, surfactant molecules were sufficiently detached away from the copolymer complexes, giving unambiguous mass spectra that were predominated by intact diblock copolymers. This ESI-based approach allowed us to investigate the relative bond strengths of metal-to-ligand complexation using collision-induced dissociation (CID) in the ion trap mass spectrometry. Conformational features and collision cross sections of the copolymers were thus obtained using subsequent ion mobility spectrometry mass spectrometry (IMS-MS). Remarkable environment-dependent conformations of the denoted diblock copolymers were found using this mass spectrometric platform.

High-energy collision-induced dissociation of radiosensitizer anions: Nimorazole and metronidazole

The formation of radical anions of nitroimidazolic radiosensitizers, nimorazole and metronidazole, and their unimolecular dissociation reactions upon high-energy collisions with He gas were investigated with a double focusing mass spectrometer equipped with an electrospray ionization (ESI) source. Radical anions of both, nimorazole and metronidazole, are readily formed in the ESI process, while for the metronidazole also the deprotonated anion is formed. A variety of dissociation channels is observed and the associated kinetic energy released in the dissociation for most of these channels is reported. The marker anion NO2− is observed in the dissociation of all studied anions. The release of NO radical from the radical anions is associated with surprisingly small kinetic energy release in comparison to simple nitroimidazoles. On the other hand, the highest kinetic energy release is noted for the release of the neutral side group from N1 position of the imidazole ring, which suggests that the substitution at the N1 plays a crucial role in the decomposition of anions of radiosensitizers.

How to run molecular dynamics simulations on electrospray droplets and gas phase proteins: Basic guidelines and selected applications.

The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.

Evaluation of 11 ionic liquids as potential extraction solvents for benzodiazepines from whole blood using liquid-liquid microextraction combined with LC-MS/MS

Ionic liquids (ILs) are becoming a trending topic in sample preparation technology, due to their low volatility, low flammability and tunable physiochemical properties. The latter affords the opportunity for creating task-specific solvents. In toxicology, these tailored ILs were not introduced yet; however, they can be significant for drug extraction purposes. This study screens 11 commercially available ILs as potential extraction media for benzodiazepines from blood samples via liquid-liquid microextraction, coupled to liquid chromatography – tandem mass spectrometry (LC-MS/MS). Structure-extraction relationships were deducted from recovery and matrix effect results (n = 5), allowing us to devise some of the favorable features in a tailored IL for benzodiazepine extraction. Overall, long alkyl chains are to be avoided, as they may sterically hinder desirable π-stacking interactions. For the same reason, aromatic planar cations, as imidazolium and pyridinium, were more effective in comparison with ammonium and pyrrolidinium. Furthermore, viscosity negatively impacted both recoveries and matrix effects. High viscosities limited mass transfer during dispersion and impeded electrospray ionization processes. In conclusion, this study evaluates which physicochemical features an IL should possess to efficiently extract benzodiazepines from whole blood.

Release of Empty Nanodiscs from Charged Droplets in the Electrospray Ionization Process: A Molecular Dynamics Study

Release of Empty Nanodiscs from Charged Droplets in the Electrospray Ionization Process: A Molecular Dynamics Study

Applying Machine Learning to Vibrational Spectroscopy.

The low-energy region of the potential energy surface (PES) of the protonated phenylalanine/serine dimer is mapped using the basin-hoping search algorithm, and 37 isomers are identified within 180 kJ·mol-1 of the global-minimum structure. Cluster structures are grouped using hierarchical clustering to partition the PES in terms of nuclear configuration. Calculated IR spectra for the various isomers are then compared with the isomer-specific IR spectra by means of the cosine distance metric to facilitate spectral assignment and identify which regions of the PES are populated in the electrospray ionization process.

Plant Metabolomics: Maximizing Metabolome Coverage by Optimizing Mobile Phase Additives for Nontargeted Mass Spectrometry in Positive and Negative Electrospray Ionization Mode.

Nontargeted screening methods with ultrahigh-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight mass spectrometry have been extensively applied to plant metabolomics to very diverse scientific issues in plant metabolomics. In this study, different mobile phase additives were tested in order to improve the electrospray ionization process and to detect as many metabolites as possible with high peak intensities in positive and negative ionization mode. Influences of modifiers were examined for nonpolar and polar compounds, as optimal conditions are not always the same. By combining different additives, metabolite coverage could be significantly increased. The best results for polar metabolites in positive ionization mode were achieved by using 0.1% acetic acid and 0.1% formic acid in negative ionization mode. For measurements of nonpolar metabolites in positive ionization mode, the application of 10 mmol/L ammonium formate led to the best findings, while the use of 0.02% acetic acid was more appropriate in negative ionization mode.

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation.

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response.