Stopped-flow electrospray ionization mass spectrometry: a new method for studying chemical reaction kinetics in solution

In this work a new mass spectrometry based method for monitoring the kinetics of chemical reactions in solution is described. A stopped-flow mixing instrument is coupled to an electrospray ionization (ESI) mass spectrometer via a novel type of interface. Chemical reactions are initiated by rapid mixing of two reactant solutions. The mixture is instantaneously transferred to a reaction tube where the kinetics can be monitored in real-time by ESI mass spectrometry. With the current setup, a time window from 2.5 to 36 seconds after mixing of the reactants can be monitored. The experimental setup is used to study the kinetics of acetylcholine hydrolysis under alkaline conditions as a function of pH. The intensities of reactant (acetylcholine) and product (choline) ions are monitored simultaneously as a function of time. The reaction is carried out under pseudo-first-order conditions and the intensity-time curves are well described by single exponentials. The rate constants determined from these fits compare favorably with previous data from the literature.     

Photosensitized oxidation of phosphatidylethanolamines monitored by electrospray tandem mass spectrometry

Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram‐negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri‐Py⁺‐Me‐PF and Tetra‐Py⁺‐Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (ESI‐MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo‐oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy‐PEs were found to be the major photo‐oxidation products. Quantification of hydroperoxides (PE‐OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE‐OOH were notorious in the presence of Tri‐Py⁺‐Me‐PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches. Copyright © 2013 John Wiley & Sons, Ltd.     

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gas- phase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation.     

Indigo Carmine degradation by hypochlorite in aqueous medium monitored by electrospray ionization mass spectrometry

The degradation of the dye Indigo Carmine by hypochlorite in aqueous solution was monitored by electrospray ionization mass spectrometry in the negative ion mode (ESI(—)‐MS). Hypochlorite was highly efficient in removing the color of aqueous solutions of the dye. ESI(—)‐MS monitoring showed that concomitant with the Indigo Carmine consumption two transient species appeared (detected as doubly charged anions) probably formed via a net insertion of two hydroxyl groups at the exocyclic C?C bond followed by the incorporation of two (mainly) or one oxygen atoms at the indolic rings of the dye. Structures of these products were proposed based on the ESI(—)‐MS/MS data and high accuracy mass measurements. These two transient intermediates quickly decomposed, both in the condensed and in the gas phase, to yield mono‐charged anions. Based on these results, a route for the Indigo Carmine degradation by hypochlorite in aqueous solution has been proposed. Copyright © 2007 John Wiley & Sons, Ltd.     

Telomestatin-induced stabilization of the human telomeric DNA quadruplex monitored by electrospray mass spectrometry

Electrospray mass spectrometry (ESI-MS) was used to monitor the kinetics of duplex formation between the human telomeric DNA quadruplex and its complementary strand; the complexation of telomestatin to the G-quadruplex delays the unwinding of the quadruplex structure and formation of the duplex.     

Observation of amide anions in solution by electrospray ionization mass spectrometry

Negative quasimolecular ions of aromatic carboxylic acid amides have been observed unexpectedly under electrospray ionization conditions. Hypothetically, deprotonation of either carboxamide or carboximidic acid tautomers can produce anions with equivalent resonance structures, the stability of which is affected by conjugated aromatic substituents. In this study, a series of meta and para substituted benzamides were analyzed using electrospray ionization mass spectrometry in aqueous methanolic solutions. The degree of ionization was found to be pH dependent and was enhanced by electron-withdrawing substituents and suppressed by electron-donating groups. The observed effect on apparent acidity can be accounted for by resonance stabilization.     

Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry 1997, 36, 12296–12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.     

Solid-state glycation of beta-lactoglobulin monitored by electrospray ionisation mass spectrometry and gel electrophoresis techniques

Glycation of beta-lactoglobulin (beta-Lg) with either lactose or galactose in a solid-state medium was monitored using gel electrophoresis techniques and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The kinetics of glycation monitored by SDS polyacrylamide gel electrophoresis showed a molecular weight increase over time of the beta-Lg bands for both sugars, but no significant amounts of aggregated proteins were observed. The isoelectric point of the protein, observed by isoelectric focusing gel electrophoresis, was dramatically affected by galactosylation. LC/MS measurements of beta-Lg variants A and B, over the whole glycation reaction time, showed a larger extent of glycation with galactose (from 4 up to 22 adducts) as compared with lactose (from 0 up to 14 adducts), and confirmed that early Maillard reaction products were the main species observed. Based on the relative abundances obtained from the deconvoluted mass spectra after a 8 h 15 min incubation time at 60 degrees C, the mean values of lactose and galactose molecules bound to the protein species were calculated to be 10.4 and 17.9, and 10.5 and 18.6, for variants A and B, respectively. Furthermore, the charge state distribution data obtained by ESI-MS was studied using different methanol percentages, and indicated that adduct formation with lactose, but more significantly galactose, tends to improve the stability properties of the native protein towards denaturation.     

Formation of gas-phase clusters monitored during electrospray mass spectrometry: a study of quaternary ammonium pesticides

Gas-phase cluster formation between the quaternary ammonium pesticides paraquat, diquat, difenzoquat, chlormequat and mepiquat, and chloride and acetate anions present in a liquid chromatography (LC) mobile phase, has been studied using electrospray mass spectrometry. The clusters of paraquat, mepiquat and chlormequat were revealed over the entire m/z range of the mass spectrometer, and their formation is dependent on the concentrations of both the cationic and the anionic species. Mepiquat and chlormequat form clusters of the type [2M(q)(+) + A(-)](+), where M(q)(+) is the quaternary ammonium cation and A(-) is the anion. Paraquat forms a cluster species with ammonia and also an ion-pair complex with chloride anions. Diquat and difenzoquat did not form observable ion-pair complexes or clusters with any of the anions studied. Competitive binding of acetate and chloride anions reflects the higher charge density of chloride, which forms the dominant clusters with mepiquat and chlormequat. The formation of cluster species has implications for the quantification of quaternary ammonium pesticides and may have an influence on the linearity of calibrations.     

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.     

Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters

Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equilibria in aqueous solution. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the solution flow rate (F(S)), the nebulizer gas flow rate (F(G)), the sprayer potential (E), and the temperature of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples containing aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. F(S) and F(G) affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra.The correlations observed between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decomposition of the dimers and of one of the monomeric complexes in the gas phase. Our results show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solutions are studied by ESI-MS.     

Observation of micelle solution of decyltrimethylammonium bromide by electrospray ionization mass spectrometry

A micelle solution of decyltrimethylammonium bromide (DTAB) was analyzed by electrospray ionization mass spectrometry. Finding an appropriate range of a capillary-skimmer potential was a prerequisite for obtaining a satisfactory spectrum. The mean molecular weight of DTAB aggregates, 10,500, was deduced from a series of mass spectra acquired at different capillary-skimmer potentials. The value was comparable with the micelle weight, previously determined by the light-scattering method.     

Characterization of a novel microperoxidase from Marinobacter hydrocarbonoclasticus by electrospray ionization tandem mass spectrometry

Microperoxidases are small heme-peptides obtained by proteolytic digestion of cytochrome c, exhibiting peroxidase activity. They consist of a short- or medium-length polypeptide chain, covalently linked to an iron protoporphyrin IX moiety via two thioether bonds involving Cys residues at the c-porphyrin A and B pyrrole rings. These small molecules are interesting for a wide range of possible applications. We have structurally characterized, by means of electrospray ionization (ESI) mass and tandem mass spectrometric experiments, a novel microperoxidase called MMP-5 (Marinobacter MicroPeroxidase-5), obtained by proteolytic digestion of cytochrome c552, a monoheminic electron-transfer protein isolated from Marinobacter hydrocarbonoclasticus. This microperoxidase, which still maintains the functional peptide moieties for peroxidase activity, is devoid of the two amino acids intercalating the Cys residues linked to the c-porphyrin, thus increasing its water solubility. Once submitted to the ESI source potential, MMP-5 showed an interesting tendency for the reduction of the iron protoporphyrin substructure. This behaviour was clearly evidenced by the mass shift exhibited by the reduced form.     

Glycoprotein profiling by electrospray mass spectrometry

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHalpha (ovine luteinizing hormone, alpha-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn(56) site of oLHalpha. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn(56) site of oLHalpha was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.     

Photolytic degradation of the insecticide thiamethoxam in aqueous medium monitored by direct infusion electrospray ionization mass spectrometry

Photodegradation of the insecticide thiamethoxam (1), 3-[(2-chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imine, in an aqueous medium was monitored by electrospray ionization mass spectrometry in the positive ion mode, ESI(+)-MS. An aqueous solution of (1) was incessantly exposed to a UV radiation source and aliquots were taken after reaction times of 1, 2, 3, and 4 h. Analysis by GC/NCI-MS revealed that (1) was continuously degraded under these experimental conditions. However, the total organic carbon (TOC) content remained practically constant during the exposition period, thereby indicating that 1 was not mineralized but continuously converted into other compounds. ESI(+)-MS monitoring revealed that whereas the intensity of the ions of m/z 292/294 ([1 + H](+)) constantly decreased, there was the emergence of other ions of m/z 247/249, 197, 168, and 116 whose intensities simultaneously increased. Their structures were proposed on the basis of: (1) the data of their ESI(+)-MS/MS; (2) their high resolution m/z values; and (3) a plausible reactivity of the thiamethoxam molecule exposed to UV radiation in aqueous solution. Finally, these data allowed us to suggest a reaction route for the photodegradation of 1 in an aqueous medium.     

Characterization of novel hydrolysis products of carbapenems by electrospray ionization mass spectrometry

Carbapenems, including meropenem and imipenem, exhibit low stability against acid or base reagents. The fragmentation behavior of meropenem and its acid hydrolysis products was investigated by Fourier transform ion cyclotron resonance electrospray ionization tandem mass spectrometry and ion trap tandem multi‐stage mass spectrometry in both positive and negative ion mode. Only one neutral loss of CO₂ was observed from the precursor ion to the MS⁴ product ions for the acid hydrolysis product and this behavior did not correspond to that expected for the previously accepted 1‐pyrroline or 2‐pyrroline structure with two carbonyl acid units. The unknown product was then proposed to be 2‐(4‐(5‐(dimethylcarbamoyl)pyrrolidin‐3‐ylthio)‐5‐imino‐3‐methyl‐6‐oxotetrahydro‐2H‐pyran‐2‐yl)‐3‐hydroxybutanoic acid on the basis of the multi‐stage mass spectrometric and accurate mass data. A similar acid hydrolysis product of imipenem was also identified by mass spectrometry, confirming that these carbapenems had the same acid hydrolysis behavior. The proposed structures were further confirmed by NMR experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: correlation between ion intensity and acid-base equilibria in solution

Electrospray ionization mass spectrometry (ESI-MS) is increasingly used in the study of metal-ligand equilibria in aqueous solutions. However, the correlation between conditions in solution and mass spectra in the gas phase is far from being completely established. In the present work the equation i = kC(0)f was used to correlate relative ion intensity (i) in an ESI mass spectrum, the stoichiometric concentration (C(0)) in solution of the complex which produced this ion, and the fraction (f) of complex having the same protonation state as that of the ion detected in the spectrum. This equation takes into account that metal-ligand complexes have acid-base properties, and that these properties affect the efficiency by which the ions are brought from the solution to the gas phase. The equation was experimentally checked by electrospraying solutions containing aluminium(III) and any of the four ligands 3,4-dihydroxybenzoic acid, 3-hydroxy-2-(1H)pyridinone, citric acid, and ethylenediaminetetramethylenephosphonic acid at different pH values. ESI-MS experimental i values and C(0)f values calculated from literature data were plotted versus the solution pH. Values are correlated in the majority of cases, thus confirming the validity of the approach proposed. Correlation is lost, as expected, for low f or C(0) values, and when extensive gas-phase reactions occur. The equation i = kC(0)f can be used to estimate quantitative data for unknown metal-ligand solutions analyzed by ESI-MS.     

A novel approach for coincidence ion mass spectrometry

A novel approach is proposed for extracting a maximum of information from secondary ions ejected when surfaces are bombarded with keV mono or polyatomic ions. It is known that the event-by-event bombardment-detection mode allows identification of spatiotemporal relationships among individual secondary ions which in turn reveal surface composition within nanometric dimensions. We have devised a procedure for identifying spatiotemporal relationships among individual secondary ions without the requirement of pulsed sample interrogation (one single projectile at a time). The consequence of "mass separated time-of-flight mass spectrometry" is a much improved measurement duty cycle.     

Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry

Recently, it has been proposed (M. Claeys et al., Science 2004; 303: 1173) that the atmospheric OH-radical-mediated photooxidation of isoprene is a source of two major secondary organic aerosol (SOA) components, that is, 2-methylthreitol and 2-methylerythritol. These diastereoisomeric tetrols, which were characterized for the first time in the fine size fraction (<2.5 microm aerodynamic diameter) of aerosols collected in the Amazon rain forest during the wet season, were proposed to enhance the capability of the aerosols to act as cloud-condensation nuclei. In the present study, we performed the oxidation of isoprene in aqueous solution under conditions that attempted to mimic atmospheric OH-radical-induced photooxidization, and monitored and characterized on-line the reaction products via electrospray ionization mass (and tandem mass) spectrometry in the negative ion mode. The results show that the reaction of isoprene with photo- or chemically generated hydroxyl radicals indeed yields 2-methyltetrols. Other polyols were also detected, and they may therefore be considered as plausible SOA components eventually formed in normal or more extreme OH-radical-mediated photooxidation of biogenic isoprene.     

Determination of micelle mass by electrospray ionization mass spectrometry

By electrospray ionization (ESI) mass spectrometry, micelle solutions of sodium cholate were investigated in detail in the presence and absence of ethanol. The average aggregation number could be evaluated from the spectra acquired under conditions where soft collisions adequate to measure the micelle solution were induced, and the value agreed well with that obtained previously by other methods. From the dependence on ethanol content, it was also found that the average aggregation number in aqueous solution without organic solvent could be reliably estimated. The ESI method proved to be a useful tool for determining the micelle mass in the original aqueous phase.