Identification of the hydrolysis products of AlCl3.6H2O by electrospray ionization mass spectrometry

The hydrolysis reactions of AlCl3 in 0.1 M aqueous solutions at pH 3.27-4.20 were monitored by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS) as a function of time. The cationic and anionic ESI mass spectra of aluminum(III) solutions gave strong evidence of the presence of a variety of monomeric and polymeric complexes. Competition between the OH- and Cl- ligands within the same aluminum core was observed. The influence of the sample cone voltage on the product distribution was also explored. The optimum sample cone voltage for the cationic spectra was 70 V. For the anionic spectra no optimum sample cone voltage was found and the appearance of the anionic spectra was strongly dependent on the sample cone voltage within the whole range explored.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Electrospray ionization tandem mass spectrometric study of salt cluster ions. Part 1--investigations of alkali metal chloride and sodium salt cluster ions

Salt cluster ions of alkali metal chlorides ACl (A = Li(+), Na(+), K(+), Rb(+) and Cs(+)) and sodium salts NaB (B = I(-), HCOO(-), CH(3)COO(-), NO(2)(-), and NO(3)(-)), formed by electrospray ionization, were studied systematically by mass spectrometry. The influences on the total positive ion and negative ion currents of variation of solvent, solution concentration, desolvation temperature, solution flow-rate, capillary voltage and cone voltage were investigated. Only cone voltage was found to influence dramatically the distribution of salt cluster ions in the mass spectra observed. Under conditions of normal cone voltage of approximately 70 V, cluster ions having magic numbers of molecules are detected with high relative signal intensity. Under conditions of low cone voltage of approximately 10 V, the distribution of cluster ions detected is characterized by a relatively low average mass/charge ratio due to the presence of multiply charged cluster ions; in addition, there is a marked reduction in cluster ions having a magic number of molecules. Product ion mass spectra obtained by tandem mass spectrometry of cluster ions are characterized by a base peak having a magic number of molecules that is less than and closest to the number of molecules in the precursor ion. Structures have been proposed for some dications and some quadruply charged ions. At pH 3 and 11, the mass spectra of NaCl clusters show the presence of mixed clusters of NaCl with HCl and NaOH, respectively. The effects of ionic radius on 20 distributions of cluster ions for 10 salts were investigated; however, the fine structure of these effects is not readily discerned.     

Electron ionisation and electrospray ionisation mass spectrometric study of a series of isomeric methyl-, dimethyl- and trimethylalloxazines

Electron ionisation (EI) mass spectra and electrospray ionisation (ESI) mass spectra at different cone voltages of a series of isomeric methyl- and dimethylalloxazines are discussed, and compared with those of lumichrome, and 1- and 3-methyllumichrome. Examination of ESI mass spectra taken at a higher cone voltage and the use of isotope-labelled methanol allow us to discuss the fragmentation pathways of [M+H]+ and [M-H](-) ions. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed in EI-MS are compared with published data. The influence of methyl and dimethyl substituents in the benzene ring on the fragmentation pathways leading to the loss of 43 and 45 Da upon both electron and electrospray ionisation is described.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrospray ionization-mass spectrometry characterization of heterotetrameric sarcosine oxidase

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i. e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.     

Effects of sample properties and mass spectroscopic parameters on electrospray ionization mass spectra of size-fractions from a soil humic acid

Size-fractions from a soil humic acid were separated by preparative size-exclusion chromatography (SEC), desalted, and concentrated by ultrafiltration and vacuum centrifugation without being subjected to any freeze-drying process. After having assessed the lack of formation of any multiple-charged ions by high-resolution Fourier transform ion cyclotron resonance electrospray ionization (ESI) mass spectrometry (MS), the size-fractions were used by direct infusion to compare the molecular ion distribution by both atmospheric pressure chemical ionization (APCI)- and ESI-MS in negative mode. The weight- (Mw) and number-averaged (Mn) molecular weight obtained by ESI-MS were invariably larger than by APCI-MS for all size-fractions, thereby indicating that ESI is more efficient than APCI to evaluate the molecular mass distribution of humic samples. No substantial difference was observed when concentration and pH of unfreeze-dried humic size-fractions were varied. The negative mode was applied to assess the effect of cone voltage from −20 to −60 V on ESI of the humic size-fractions further separated through an on-line SEC column. The resulting mass spectra and Mw and Mn values suggested that the variation of cone voltage in ESI-MS affects the ionization potential of associated humic molecules more in solution rather than their fragmentation. These findings agree with previous observations which indicated a limitation of ESI in providing consistent mass detection for a complex mixture of heterogeneous humic molecules, especially when they are aggregated by a freeze-drying process.     

Electron ionization and electrospray mass spectra of diaryl-substituted enaminoketones and their thio analogs

Electron ionization (EI) and positive electrospray ionization (ESI) mass spectra of selected diaryl enaminoketones and enaminothiones have been studied. In the EI mass spectra of both classes of compound, molecular ion peaks are accompanied by the peaks corresponding to the [M-H](+) ions. The formation of these ions can be rationalized by a cyclization reaction resulting in the formation of the respective isoxazolium and isothiazolium cations. Under positive ESI conditions, in the spectra recorded for the enaminoketones peaks corresponding to the [M+H](+), [M+Na](+) and [2M+Na](+) ions appeared, while in the spectra recorded for the enaminothiones, peaks corresponding to the [M-H](+) ions were dominant. These ions are most likely formed by oxidation of the neutral enaminothione molecules on the surface of the positively charged stainless steel capillary in the ESI ion source (anodic oxidation).     

Solution or Gas Phase? Oxidation and Radical Formation in Electrospray Ionization Mass Spectrometry (ESI MS)

The relationship between one‐electron (e^?) oxidation processes and the formation of radical cations of endogenous and exogenous compounds in vivo is of considerable interest. This paper reports on the experiments that allow FTICR mass spectrometric (MS) detection of ion signals that are consistent with the formation of radical cations of caffeine (CA) and theophylline (TP) during electrospray ionization (ESI) in ESI FTICR MS and in on‐line electrochemistry (EC)/ESI FTICR MS in positive mode. Significantly, the signals of the radicals of CA^?+ and TP^?+can be enhanced by simple modifications of the operating conditions in ESI MS, facilitating investigations of radical formation and related reactions.     

Precision profiling and identification of human serum peptides using Fourier transform ion cyclotron resonance mass spectrometry

Many biomarker discovery studies are based on matrix-assisted laser desorption/ionisation (MALDI) peptide profiles. In this study, 96 human serum samples were analysed on a Bruker solariX(TM) MALDI Fourier transform ion cyclotron resonance (FTICR) system equipped with a 15 tesla magnet. Isotopically resolved peptides were observed in ultrahigh resolution FTICR profiles up to m/z 6500 with mass measurement errors (MMEs) of previously identified peptides at a sub-ppm level. For comparison with our previous platform for peptide profile mass analysis (i.e. Ultraflex II) the corresponding time-of-flight (TOF) spectra were obtained with isotopically resolved peptides up to m/z 3500. The FTICR and TOF systems performed rather similar with respect to the repeatability of the signal intensities. However, the mass measurement precision improved at least 10-fold in ultrahigh resolution data and thus simplified spectral alignment necessary for robust and quantitatively precise comparisons of profiles in large-scale clinical studies. From each single MALDI-FTICR spectrum an m/z-list was obtained with sub-ppm precision for all different species, which is beneficial for identification purposes and interlaboratory comparisons. Furthermore, the FTICR system allowed new peptide identifications from collision-induced dissociation (CID) spectra using direct infusion of reversed-phase (RP) C(18)-fractionated serum samples on an electrospray ionisation (ESI) source.     

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.     

Effect of solution acidity on cytochrome c conformations of alternating current electrospray ionization mass spectrometry

This paper reports notable observations regarding the ion charge states of thermally stable cytochrome c, generated using an alternating current (AC) electrospray ionization (ESI) device. An AC ESI sprayer entrains low-mobility ions to accumulate at the meniscus cone tip prior to the ejection of detached aerosols to produce analyte ions. Therefore, as the solvent acidity varies, protein ions entrained in the AC cone tip are found to change conformation less significantly compared with those in the direct current (DC) cone. We acquired the AC ESI mass spectra of cytochrome c at pH range from 2 to 4. Unlike the DC ESI mass spectra showing clear conformation changes due to denaturing, the AC spectra indicated that only partial denaturing occurs even at extremely acidic pH 2. More native cytochrome c in lower charge states therefore remained. Moreover, with a solvent mixture of aqueous buffer and acetonitrile (70:30), partially denatured cytochrome c was still preserved at pH 2 by using AC ESI. Completely denatured proteins are observed at pH 2 by using DC ESI.     

Free energy for blue copper protein unfolding determined by electrospray ionisation mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determination of the free energy (DeltaG) of unfolding of proteins is described. The method was tested using three blue copper proteins: wild type azurin, Cys-3Ala/Cys-26Ala (C3A/C26A) azurin mutant and wild-type amicyanin. The time course of the denaturation process of the proteins dissolved in methanol/water (50:50, v/v, pH 3.5) was followed by recording ESI mass spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the free energy for the unfolding process for the three proteins could be determined. To evaluate the reliability of the thermodynamic data obtained by the ESI mass spectrometric approach, the denaturation process was followed by UV-VIS spectroscopy. The two sets of data obtained by these independent methods were in good agreement indicating that the ESI-MS approach can be used to obtain reliable quantitative information about the protein unfolding process. In principle, this approach can be applied to other proteins and requires very low amounts of sample, due to the intrinsic sensitivity of mass spectrometry. This may prove particularly useful when the amount of sample available prevents the use of current methods.     

External accumulation of ions for enhanced electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.     

Electrospray tandem quadrupole fragmentation of quinolone drugs and related ions. On the reversibility of water loss from protonated molecules

Selected reaction monitoring (SRM) of quinolone drugs showed different sensitivities in aqueous solution vs. biological extract. The authors suggested formation of two singly protonated molecules with different behavior, one undergoing loss of H(2)O and the other loss of CO(2), so that SRM transitions might depend on the ratios of these forms generated by the electrospray. These surprising results prompted us to re-examine several quinolone drugs and some simpler compounds to further elucidate the mechanisms. We find that the relative contributions of loss of H(2)O vs. loss of CO(2) in tandem mass spectrometric (MS/MS) experiments depend not only on molecular structure and collision energy, but also, in certain cases, on the cone voltage. We further find that many product ions formed by loss of H(2)O can reattach a water molecule in the collision cell, whereas ions formed by loss of CO(2) do not. Since reattachment of H(2)O can occur after water loss in the cone region and prior to selection of the precursor ion, this effect leads to the dependence of MS/MS spectra on the cone voltage used in creating the precursor ion, which explains the formerly observed effect on SRM ratios. Our results support the earlier conclusion that varying amounts of two ions of the same m/z value are responsible for problems in the analysis of these drugs, but the origin is in dehydration/rehydration reactions. Thus, SRM transitions for certain complex compounds may be comparable only when monitored under equivalent ion-forming conditions, including the voltage used in the production of the protonated molecules in the electrospray ionization (ESI) source.     

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐ tandem mass spectrometry

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.     

Synchronized dual-polarity electrospray ionization mass spectrometry

This work describes the synchronized dual-polarity (DP) electrospray ionization (ESI) method and demonstrates the first DP ESI mass spectra obtained using two mass spectrometers. Stable double Taylor cones were produced by applying two counter electric voltages with opposite polarities to one electrosprayer. The development of double Taylor cones required higher extraction voltages than conventional ESI, but DP ESI worked effectively at liquid flow rate range three times wider than conventional ESI. Using pure methanol, the emission currents of the two cones were neutralized and no current was drawn from the sprayer. Synchronized DP mass spectra were obtained using electrospray calibrants dissolved in methanol solution of low water content. For bovine insulin with conventional electrospray solution, the gas-assisted electrospray delivered satisfactory sensitivity and stability for routine mass analyses.     

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.     

Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.