Electrospray Ionization Experiments Research Articles

Mass spectrometry study of ascorbyl palmitate as an agent for nanosomes formation

Background. Study of properties and intermolecular interactions of biologically active compounds which can be used for the purposes of transmembrane drugs delivery is a topical task of modern molecular biophysics. Ascorbyl Palmitate (AP) as a fat-soluble form of vitamin C has recently attracted attention as a promising agent for formation of nanosomes for the “fat insoluble” drug molecules transfer through membranes. However, AP is not sufficiently characterized by up-to-date soft ionization mass spectrometric techniques. Objectives. The aim of the present work is to characterize AP and its intermolecular interactions by a number of mass spectrometric techniques: Electrospray Ionization (ESI), Laser Desorption/Ionization (LDI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). The comparison of these techniques applicability to the study of AP intermolecular interactions as a drug delivery assisting agent is scheduled. Methods. ESI mass spectra are obtained with triple quadrupole Micromass Quattro mass spectrometer. LDI and MALDI experiments are performed by Autoflex II mass spectrometer. Results. In the ESI experiments in the positive ion mode abundant peaks of protonated and cationized AP molecules as well as the peaks of AP clusters nAP•H+ and nAP•Na+ (n=2÷4) are revealed in the mass spectra. This result testifies to the formation of stable noncovalent complexes of the AP molecules in the polar media and confirms the AP ability of formation nanosomes for drug delivery. Analysis of LDI and MALDI mass spectra of AP in positive and negative ion modes shows that in the presence of molecular ions of AP, the peaks of AP dimers or larger AP clusters are not recorded. The ESI probing of the model system containing AP and dipalmitoylphosphatidylcholine (DPPC) reveals stable AP•DPPC•H+ complex which models the AP intermolecular interactions with the phospholipid components of biomembranes and/or liposomes under AP functioning as a drug delivery assisting agent. Conclusions. The current study demonstrates the applicability of all tested mass spectrometric techniques for AP identification in solutions and solid phase, while for the purpose of examining of the AP noncovalent complexes formation and study of AP interactions with biomolecules the ESI is defined as the most effective technique.

Using Density Functional Theory for Testing the Robustness of Mobile-Proton Molecular Dynamics Simulations on Electrosprayed Ions: Structural Implications for Gaseous Proteins.

Current experiments only provide low-resolution information on gaseous protein ions generated by electrospray ionization (ESI). Molecular dynamics (MD) simulations can yield complementary insights. Unfortunately, conventional MD does not capture the mobile nature of protons in gaseous proteins. Mobile-proton MD (MPMD) overcomes this limitation. Earlier MPMD data at 300 K indicated that protein ions generated by "native" ESI retain solution-like structures with a hydrophobic core and zwitterionic exterior [Bakhtiari, M.; Konermann, L. J. Phys. Chem. B 2019, 123, 1784-1796]. MPMD redistributes protons using electrostatic and proton affinity calculations. The robustness of this approach has never been scrutinized. Here, we close this gap by benchmarking MPMD against density functional theory (DFT) at the B3LYP/6-31G* level, which is well suited for predicting proton affinities. The computational cost of DFT necessitated the use of small peptides. The MPMD energetic ranking of proton configurations was found to be consistent with DFT single-point energies, implying that MPMD can reliably identify favorable protonation sites. Peptide MPMD runs converged to DFT-optimized structures only when applying 300-500 K temperature cycling, which was necessary to prevent trapping in local minima. Temperature cycling MPMD was then applied to gaseous protein ions. Native ubiquitin converted to slightly expanded structures with a zwitterionic core and a nonpolar exterior. Our data suggest that such inside-out protein structures are intrinsically preferred in the gas phase, and that they form in ESI experiments after moderate collisional excitation. This is in contrast to native ESI (with minimal collisional excitation, simulated by MPMD at 300 K), where kinetic trapping promotes the survival of solution-like structures. In summary, this work validates the MPMD approach for simulations on gaseous peptides and proteins.

Compound annotation in liquid chromatography/high-resolution mass spectrometry based metabolomics: robust adduct ion determination as a prerequisite to structure prediction in electrospray ionization mass spectra.

A bottleneck in metabolic profiling of complex biological extracts is confident, non-supervised annotation of ideally all contained, chemically highly diverse small molecules. Recent computational strategies combining sum formula prediction with in silico fragmentation achieve confident de novo annotation, once the correct neutral mass of a compound is known. Current software solutions for automated adduct ion assignment, however, are either publicly unavailable or have been validated against only few experimental electrospray ionization (ESI) mass spectra. We here present findMAIN (find Main Adduct IoN), a new heuristic approach for interpreting ESI mass spectra. findMAIN scores MS1 spectra based on explained intensity, mass accuracy and isotope charge agreement of adducts and related ionization products and annotates peaks of the (de)protonated molecule and adduct ions. The approach was validated against 1141 ESI positive mode spectra of chemically diverse standard compounds acquired on different high-resolution mass spectrometric instruments (Orbitrap and time-of-flight). Robustness against impure spectra was evaluated. Correct adduct ion assignment was achieved for up to 83% of the spectra. Performance was independent of compound class and mass spectrometric platform. The algorithm proved highly tolerant against spectral contamination as demonstrated exemplarily for co-eluting compounds as well as systematically by pairwise mixing of spectra. When used in conjunction with MS-FINDER, a state-of-the-art sum formula tool, correct sum formulas were obtained for 77% of spectra. It outperformed both 'brute force' approaches and current state-of-the-art annotation packages tested as potential alternatives. Limitations of the heuristic pertained to poorly ionizing compounds and cationic compounds forming [M]+ ions. A new, validated approach for interpreting ESI mass spectra is presented, filling a gap in the nontargeted metabolomics workflow. It is freely available in the latest version of R package InterpretMSSpectrum.

Charge-induced instabilities of droplets containing macromolecular complexes

Solvated macromolecular complexes are ubiquitous in nature, notably in biological systems containing proteins and nucleic acids. Studies of the interactions within a macromolecular complex and between the complex and the solvent in droplet environments are critical for understanding the stability of macromolecular complexes in electrospray ionization (ESI) and nanofluidic experiments. In this study, two distinct cases of macromolecular complexes in aqueous nanodrops are examined by using molecular dynamics simulations: (i) a pair of sodiated poly(ethylene) glycol (PEG) macroions and (ii) a double-stranded DNA (dsDNA). PEG represents a case in which the surface energy of the aqueous droplet is larger than the solvent–macromolecule energy. Conversely, in a droplet solvating dsDNA, the solvent–macromolecule interaction energy overcomes the solvent interaction energy. We report that charge-induced instabilities previously identified for single macroions also appear in the case of complexes, but with a higher level of complexity. In the case of a pair of PEG macroions, we found that their conformations on the surface of a droplet “sense” each other. The charged PEGs are each released from a droplet at different times through contiguous extrusion or drying-out mechanisms. In the case of the DNA, the charge-induced instability manifests as a spine droplet morphology. Narrow regions of the spines promote break down of the hydrogen bonds that hold the dsDNA together. The dsDNA separates into two single strands as it is increasingly exposed to vacuum. These findings elucidate charge-induced instabilities of macromolecular complexes in droplets, which are critical intermediates in ESI and nanofluidic experiments.

Laser-Induced Azomethine Ylide Formation and Its Covalent Entrapment by Fulleropyrrolidine Derivatives During MALDI Analysis

Two novel monofunctionalized fulleropyrrolidine derivatives (Prato adducts) were prepared and characterized by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI experiments conducted in the positive-ion mode on pure and mixed samples of both monofunctionalized fullerene derivatives revealed the efficient formation of bisadducts (in the case of the pure samples) and mixed bisadducts (in the case of a mixed sample). Bisadducts were not observed in the ESI experiments and thus not present in the sample. A mechanism for the MALDI formation of these bisadduct ions is proposed in which an azomethine ylide fragment is formed in situ from the monofunctionalized fulleropyrrolidine species upon laser irradiation. This fragment, which can survive as an intact moiety in the gas phase in the special environment provided by the MALDI experiment, is then able to attach to a fulleropyrrolidine monoadduct which acts as a dipolarophile, thus leading to the formation of a bisadduct fullerene derivative. The unprecedented re-attachment of the azomethine ylide implies that the establishment of the ligand attainment of Prato adducts based on MALDI analysis alone can lead to wrong assignments.

Novel product ions of 2-aminoanilide and benzimidazole Ag(I) complexes using electrospray ionization with multi-stage tandem mass spectrometry

The 2-aminoaniline scaffold is of significant value to the pharmaceutical industry and is embedded in a number of pharmacophores including 2-aminoanilides and benzimidazoles. A novel application of coordination ion spray mass spectrometry (CIS-MS) for interrogating the silver ion (Ag(+)) complexes of a homologous series of these compounds using multi-stage tandem mass spectrometry is described. Unlike the ubiquitous alkali metal ion complexes, Ag(+) complexes of 2-aminoanilides and benzimidazoles were found to yield [M - H](+) ions in significant abundance via gas-phase elimination of the metal hydride (AgH) resulting in unique product ion cascades. Sample introduction was by liquid chromatography with mass spectrometry analysis performed on a hybrid linear ion trap/orbitrap instrument capable of high-resolution measurements. Rigorous structural characterization by multi-stage tandem mass spectrometry using [M + H](+), [M - H](-) and [M - H](+) precursor ions derived from ESI and CIS experiments was performed for the homologous series of 2-aminoanilide and benzimidazole compounds. A full tabular comparison of structural information resulting from these product ion cascades was produced. Multi-stage tandem mass spectrometry of [M - H](+) ions resulting from Ag(+) complexes of 2-aminoanilides and benzimidazoles in CIS-MS experiments produced unique product ion cascades that exhibited complementary structural information to that obtained from tandem mass spectrometry of [M + H](+) and [M - H](-) ions by electrospray ionization (ESI). These observations may be broadly applicable to other compounds that are observed to form Ag(+) complexes and eliminate AgH.

Characterization of Ce3+-tributyl phosphate coordination complexes produced by fused droplet electrospray ionization with a target capillary

Coordination complexes containing Ce(III) and tri-n-butyl phosphate (TBP) in the 1+, 2+ and 3+ charge states were generated using both direct infusion electrospray ionization (ESI) and fused droplet (FD) ESI using a target capillary, in which the analyte solutions are impinged by the ESI droplets. The same coordination complexes were produced in each experiment, and their relative abundances were also very close, suggesting that similar processes are occurring in both experiments. The ion species formed in both experiments have the general formula [Ce(NO(3) )(m=0-2) (TBP)(n=3-7) ]((3-m)+) . The appearance of abundant 1+ and 2+ ion pair complexes indicated that the ESI process was modifying the ion populations in the original solutions, which contain predominantly 3+ and 2+ species. The FD ESI experiments were less sensitive for coordination complexes compared to direct infusion ESI; however, mid-picomolar quantities of coordination complexes were measured using the target capillary, indicating that sensitivity would be sufficient for measuring species in many industrial separations processes.

Peptide polarity and the position of arginine as sources of selectivity during positive electrospray ionisation mass spectrometry

Electrospray ionisation (ESI) is a selective process and, for similar sized analytes, the intrinsic properties of the molecules affect the ionisation process and their response. This research sets out to determine the effect of some of these properties in peptides: peptide polarity and the presence of arginine at positions 1 and 4 in the amino acid sequence on the ESI response. Six peptides; molecular mass ranges 1.3-1.6 kDa; substance P (SP) and glutamate fibrinopeptide (GFP) and 3.2-3.7 kDa; calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), glucagon-like peptide 1 (GLP1) and defensin human neutropeptide 2 (DHNP2), were investigated. We have demonstrated that in positive ESI, for similar sized peptides and the same charge state, the responsiveness is in the order: Peptides with N or C terminal arginine > most non-polar peptides > least non-polar peptides. Therefore, arginine at the terminal position is a source of selectivity. Data from matrix-assisted laser desorption ionisation (MALDI) analysis supports that of the ESI experiments: Peptides with a terminal arginine residue generated higher signal intensities. Our observations extend our understanding of the ESI process and provide a rational approach to optimising sensitivity of electrospray conditions where a narrow mass range of peptides are poorly chromatographically resolved. This information will provide for a more effective method development process, especially during label-free quantitative determination of peptides extracted in solution.

How Are Completely Desolvated Ions Produced in Electrospray Ionization: Insights from Molecular Dynamics Simulations

We apply molecular dynamics (MD) simulations to study the final phase of electrospray ionization (ESI), where an ion loses all of its associated solvent molecules. By applying an electric field to a cluster of H(2)O molecules solvating an ion and including a surrounding gas of varying pressure, we demonstrate that collisions with the gas play a major role in removing this final layer of solvent. We make quantitative predictions of the critical velocity required for the cluster to start losing molecules via collisions with gas and propose that this should be important in real ESI experiments. Such collisions have heretofore not been explicitly considered in discussions of the ESI process.

Anion Effects on Sodium Ion and Acid Molecule Adduction to Protein Ions in Electrospray Ionization Mass Spectrometry

Gaseous protein-metal ion and protein-molecule complexes can be readily formed by electrospray ionization (ESI) from aqueous solutions containing proteins and millimolar concentrations of sodium salts of various anions. The extent of sodium and acid molecule adduction to multiply charged protein ions is inversely related and depends strongly on the proton affinity (PA) of the anion, with extensive sodium adduction occurring for anions with PA values greater than ~300 kcal·mol(-1) and extensive acid molecule adduction occurring for anions with PA values less than 315 kcal·mol(-1). The role of the anion on the extent of sodium and acid molecule adduction does not directly follow the Hofmeister series, suggesting that direct protein-ion interactions may not play a significant role in the observed effect of anions on protein structure in solution. These results indicate that salts with anions that have low PA values may be useful solution-phase additives to minimize nonspecific metal ion adduction in ESI experiments designed to identify specific protein-metal ion interactions.

Electrosonic spray ionization—An ideal interface for high-flow liquid chromatography applications

Electrosonic spray ionization (ESSI) has been studied as an interface between high-performance liquid chromatography (HPLC) and mass spectrometry (MS), using sample flow rates up to 3.0 ml min −1. This ionization interface was compared with pneumatically assisted electrospray ionization (ESI) using mass spectrometry for detection. For experiments that did not involve direct comparison of different flow rates, the ESI experiments were performed using post column splitting to work at optimal conditions. ESSI allows the interfacing of conventional or high-resolution liquid chromatography (LC) methods to mass spectrometry without post column splitting. High sample flow rates could be handled without a significant loss of signal intensity using a nebulization gas flow rate of 5.5 L min −1. Since ESI needs to be operated with lower sample flow rates, it is limited to micro/nano LC systems, or post column splitting must be used. In particular, nano LC systems have to be treated with great care and require constant maintenance. When using post-column splitting, the increased diffusion can become a problem especially when using systems with very small void volumes. In all experiments ESSI showed better signal intensities than a commercially available, pneumatically assisted ESI source. ESSI does not require heating of the nebulizer gas, which should help to preserve the original structure of thermally unstable molecules. Therefore, ESSI is presented as an alternative to the commercially available heated ESI sources of AB SCIEX, Thermo Fischer, Agilent and Waters. The observed LC-ESSI-MS ion chromatograms are shown to be very stable even when using flow rates higher than 1.0 ml min −1, which could be very suitable for ultra high performance LC, where sample flow rates up to 2.0 mL min −1 with backpressures up to 1200 bar are used. Also, a difference in the relative intensities of singly and doubly protonated peptide monomers and dimers was observed between the two ionization methods. The coefficients of determination for the calibration of instrument response for Val–Tyr–Val and Met-Enkephalin showed excellent linearity over a wide concentration range (0.1–100 μM), while ESI results were only linear over a much smaller range (0.1–20 μM). The observed behavior is thought to be caused by insufficient ionization efficiency of solutions above ∼20 μM by ESI, exemplifying the robustness of ESSI as an interface between LC and MS.

A Corona Discharge Initiated Electrochemical Electrospray Ionization Technique

We report here the development of a corona discharge (CD) initiated electrochemical (EC) electrospray ionization (ESI) technique using a standard electrospray ion source. This is a new ionization technique distinct from ESI, electrochemistry inherent to ESI, APCI, and techniques using hydroxyl radicals produced under atmospheric pressure conditions. By maximizing the observable CD at the tip of a stainless steel ESI capillary, efficient electrochemical oxidation of electrochemically active compounds is observed. For electrochemical oxidation to be observed, the ionization potential of the analyte must be lower than Fe. Ferrocene labeled compounds were chosen as the electrochemically active moiety. The electrochemical cell in the ESI source was robust, and generated ions with selectivity according to the ionization potential of the analytes and up to zeptomolar sensitivity. Our results indicate that CD initiated electrochemical ionization has the potential to become a powerful technique to increase the dynamic range, sensitivity, and selectivity of ESI experiments.

Can electrospray ionization help the characterization of iron gall inks? Investigation on the interactions of gallic acid with iron ions in aqueous solutions

In the present work, an investigation has been conducted by electrospray ionization (ESI) experiments to characterize the structures of iron gall ink complexes in solution. Simple mono and polyphenolic acid molecules added to iron sulfate salts were chosen to model the recipes of ink composition. Theoretical calculations have been used (1) to determine the stability of the ionic complexes generated in the gas phase, (2) to explain which structures are more likely generated in the electrospray ion source, and (3) to determine which mechanisms are likely involved in their formation. Fragmentation pathways of the derived structures have also been investigated and rationalized to facilitate the interpretation of the data obtained under collisionally induced dissociation (CID) conditions.The present study confirms the assumption that ESI experiments with ions that are preformed in solution must be considered carefully. As a matter of fact, the study of ion formation mechanisms in the ion source is necessary to establish relationships between the ion structures in the condensed phase and the gas phase.

Lipids of human meibum: mass-spectrometric analysis and structural elucidation

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MS(n)). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MS(n) detection of the analytes (HPLC API MS(n)). The major precursor ions were isolated and subjected to further sequential fragmentation in MS(n) experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M - H(2)O + H](+)) and oleic acid (OA; m/z 283; [M + H](+)) were found. The other major compounds of the general molecular formula C(n)H(2n-2)O(2) were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas C(n)H(2n-4)O(2) and C(n)H(2n)O(2). Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MS(n) analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.

Biomolecule structure characterization in the gas phase using mass spectrometry

Carbohydrate/cation interactions were examined in the gas phase using mass spectrometry and the results were compared with computer generated models of the complexes. Monosaccharide/alkali cation complexes of five carbohydrates, D‐fructose, D‐glucose, D‐galactose, D‐mannose, and a deuterated analog of D‐glucose, 6,6‐D‐glucose‐d2, were studied. Among the technuques used in this effort were electrospray ionization (ESI), desorption/ionization on silicon (DIOS), matrix‐assisted laser desorption/ionization (MALDI) and fast atom/ion bombardment (FAB) mass spectrometry. A series of ESI, DIOS, MALDI and FAB‐MS experiments were used to obtain relative cation binding preferences of each monosaccharide. Heterodimers of 6,6‐D‐glucose‐d2 formed with each of the monosaccharides show that Na+ binding for D‐fructose, D‐mannose and D‐galactose is similar, while D‐glucose was 25% weaker. Modeling studies and energy minimization calculations on the alpha and beta forms of the monosaccharide alkali cation complexes are consistent with the experimental data and indicate that D‐fructose, D‐galactose, and D‐mannose undergo tridentate and tetradentate binding with Na+ and Li+ while D‐glucose would only form a bidentate complex.

Analysis of enantiomeric excess using mass spectrometry: fast atom bombardment/sector and electrospray ionization/Fourier transform mass spectrometric approaches

The utility of fast atom bombardment (FAB) ionization on a sector mass spectrometer, and of electrospray ionization (ESI) on a Fourier transform ion cyclotron resonance mass spectrometer, for enantiomeric excess measurements was explored. Both methods involved the same host–guest system: ( R, R)- or ( S, S)-dimethyldiketopyridino-18-crown-6 (host) and α-(1-naphthyl)ethylammonium (guest). Both use an achiral amine (benzylamine for the FAB experiments, cyclohexylamine for the ESI experiments) as an internal reference compound and involve competitive complexation of the achiral and chiral amines with the chiral host. The FAB experiments are shown to give stable, reproducible results, but exhibit a smaller degree of enantiodiscrimination than the ESI experiments. The ESI experiments, which involve measurement of apparent guest exchange equilibrium constants, show a linear relationship between apparent equilibrium constant and enantiomeric excess. The apparent equilibrium constant is shown to be a composition-weighted average of the equilibrium constants for the two pure enantiomers. Enantiomeric impurities as small as about 2% can currently be detected.