FlexESI: An Automated Vapor-Switching Interface for Enhanced Flexibility and Sensitivity in Electrospray Ionization

Characterization of ageing products of ester-based synthetic lubricants by liquid chromatography with electrospray ionization mass spectrometry and by electrospray ionization (tandem) mass spectrometry

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.

The determination of seven saponins in crude plant extracts by electrospray ionization mass spectrometry (ESI‐MS) and fast atom bombardment mass spectrometry (FAB‐MS) is described. Distinct protonated and natriated (Na‐adduct) molecular ions in ESI‐MS spectra readily provide molecular weight information, which can be further verified using clusters of molecular ions. Saponin mixtures can be analyzed by ESIMS on varying the potential difference between the capillary and skimmer in the ESI source to decompose impurities. ESI‐MS uses less amount of sample than that required by FAB‐MS. ESI‐MS does not produce structural information, however. The FAB‐MS spectra consist mainly of protonated and deprotonated molecular ions with limited structural information. (‐)‐FAB‐MS is more suitable for analyzing saponin samples than the (+)‐FAB‐MS.

Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells. We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease.

Multidimensional Mass Spectrometry Assisted Metallo-Supramolecular Chemistry

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Chapter 2 Characterization of Protein Higher Order Structure and Dynamics with ESI MS

Fourier‐transform ion cyclotron resonance mass spectrometry (MS) has demonstrated potential to revolutionize the fields of limnology and chemical oceanography by identifying the individual molecular components of organic matter in natural waters. The use of MS for this purpose is made possible by the electrospray technique which successfully ionizes polar, nonvolatile organic molecules. Another recently developed ion source, atmospheric pressure photoionization (APPI), extends MS capabilities to less polar molecules. This article presents early results on the application of APPI MS to natural organic matter. We compare APPI MS and electrospray MS data for dissolved organic matter from Lake Drummond (Virginia, USA). Collectively, electrospray and APPI MS identify more than 6000 molecular species to which we assign unique molecular formulas. Fewer than 1000 molecular species are common to both electrospray and APPI mass spectra, indicating that the techniques are highly complementary in the types of molecules they ionize. Access to a broad range of molecules provided by combining APPI and electrospray has prompted a qualitative analysis. The goal is to assess the extent to which molecular MS data correspond with elemental (CHNOS) and structural characteristics determined by combustion elemental analyses and 13C nuclear magnetic resonance (NMR). Because the data obtained by these different methods are not directly comparable, we propose a novel data analysis procedure that facilitates their comparison. The bulk elemental composition calculated from electrospray MS data are in close agreement (± 15%) with values determined by combustion elemental analysis. APPI and electrospray MS detect protein contributions in agreement with 13C NMR (6 wt %) but underestimate carbohydrates relative to 13C NMR. Nevertheless, MS results agree with NMR on the relative proportions of noncarbohydrate compounds in the organic matter: lignins > lipids > peptides. Finally, we use a molecular mixing model to simulate a 13C NMR spectrum from the MS datasets. The correspondence of the simulated and measured 13C NMR signals (74%) suggests that, collectively, the molecular species identified by APPI and electrospray MS comprise a large portion of the organic matter in Lake Drummond. These results add credibility to electrospray and APPI MS in limnology and oceanography applications, but further characterization of ion source behavior is fundamental to the accurate interpretation of MS data.

Mass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two “soft” ionization techniques: matrix-assisted laser desorption/ ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fi ssion, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).

It is frequently said that DESI-MS follows a similar ionization mechanism as ESI because of similarities usually observed in their respective mass spectra. However, practical use of DESI-MS for protein analysis is limited to proteins with lower molecular weights (< 25kDa) due to a mass-dependent loss in signal intensity. Here we investigated commonly used volatile acids and their ammonium salt buffers for DESI-MS analysis of protein. We noticed that, surprisingly, some additives influence the analysis differently in DESI compared to ESI. Improved signal intensities with both DESI and ESI were obtained when acetic and formic acid were added into aqueous methanol spray solvents with both DESI and ESI. On the other hand, while with ESI the addition of ammonium salts into spray solutions strongly reduced both signal and S/N, with DESI signal intensities and S/N were improved dramatically. Ammonium bicarbonate when used with DESI reduced the total amount of adduction and delivered excellent signal-to-noise ratios with high intensity; however, it also denatures protein. When native state protein mass spectra are preferred, ammonium acetate would also deliver reasonable adduct removal and improved S/N. The amount of total adduction of individual adducting species and of all species could not be correlated with differences in either solutions pH values or with proton affinities of the anions. An obvious difference between DESI and ESI mass spectrometry is the effects of protein solubility during droplet pickup (desorption), but differences in the sizes, velocities, and composition of ionizing droplets were also discussed as important factors. Graphical Abstract ᅟ.

Electrospray ionization mass spectrometric study of encapsulation of amino acids by cyclodextrins

The initial steps toward routinely applying mass spectrometry in the biochemical laboratory have been achieved. In the past, mass spectrometry was confined to the realm of small, relatively stable molecules; large or thermally labile molecules did not survive the desorption and ionization processes intact. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allow for the analysis of both small and large biomolecules through "mild" desorption and ionization methods. The use of ESI and MALDI mass spectrometry extends beyond simple characterization. Noncovalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery are among the areas to which ESI and MALDI mass spectrometry have been applied. This review summarizes recent developments and major contributions in mass spectrometry, focusing on the applications of MALDI and ESI mass spectrometry.

Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono- and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI-MS). Despite minor matrix-dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono- and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI-MS.

Organocuprates are important reagents with numerous applications in preparative organic chemistry, which has led to extensive investigations regarding their molecular composition. However, while lithium organocuprates have been thoroughly studied, the solution speciation of magnesium organocuprates is only poorly understood. Organocuprates have also been proposed as important intermediates in certain copper-mediated trifluoromethylation reactions, but their origin as well as the overall reaction mechanism remained unclear. The higher homologues – organoargentates and -aurates – have been characterized to a much lesser extent, and only little is known about their speciation and aggregation. Despite the potential of silver to promote cross-coupling reactions, the mechanisms of these reactions and the role of organoargentates in these synthetically valuable but rarely used transformations are unknown. In the present work, electrospray-ionization (ESI) mass spectrometry and collision-induced dissociation (CID) experiments were used to identify and characterize organocuprates, -argentates, and -aurates. Furthermore, the copper-mediated trifluoromethylation of terminal alkynes as well as silver-mediated cross-coupling reactions were investigated by ESI mass spectrometry and CID experiments. The results show great similarities in the speciation of solutions of organocuprates, -argentates, and -aurates that were prepared from the respective coinage-metal cyanide and organolithium reagents in tetrahydrofuran (THF). In particular, the formation of polynuclear, lithium-containing heterobimetallic organometallates was observed for all three coinage metals. These complexes closely resemble species that were detected in previous ESI mass spectrometric studies of cyanocuprates and Gilman cuprates in ethereal solvents. In contrast, organocuprates that were prepared by transmetallation of a copper precursor with Grignard reagents in THF showed a different behavior. ESI mass spectra of these reagents did not exhibit heterobimetallic cuprates in significant amounts, but were dominated by monometallic species. This difference between organocuprates prepared from organolithium or organomagnesium reagents is attributed to the formation of different lithium- and magnesium counter-ions. ESI mass spectra of organoargentate and -aurate solutions prepared from silver cyanide or gold cyanide, respectively, and Grignard reagents also showed monometallic organoargentates and -aurates, which is in agreement with the outcome of mass spectrometric analysis of the magnesium organocuprates. However, these reactions were, in part severely, affected by hydrolysis reactions. The unimolecular gas-phase reactivity of the observed organometallates was studied by CID experiments. The polynuclear, lithium-containing heterobimetallic complexes of all three coinage metals prefer similar deaggregation reactions, while the mononuclear coinage metallates show different reaction pathways that apparently depend on the actual coinage metal present in the complex. Presumably, the overall structure of the larger heterobimetallic species governs the outcome of their fragmentation reactions, while the intrinsic reactivity of the individual coinage metals becomes the determining factor in the fragmentation behavior of the mononuclear metallates. Polynuclear but monometallic cuprates that were prepared from Grignard reagents also showed deaggregation reactions. In addition to that, beta-hydride elimination and beta-methyl elimination reactions were obseverd in two cases. In order to investigate the copper-mediated trifluoromethylation of alkynes, solutions of copper(I) iodide, potassium fluoride, and trimethyl(trifluoromethyl)silane (the Ruppert-Prakash reagent) were examined with and without the addition of a terminal alkyne. In the absence of an alkyne component, homoleptic trifluoromethyl cuprates could be identified in ESI mass spectra of these solutions. The addition of terminal alkynes additionally afforded heteroleptic trifluoromethyl cuprates bearing three trifluoromethyl groups and an alkynyl ligand. In gas-phase fragmentation experiments, these key copper(III) intermediates underwent reductive eliminations that led to the exclusive release of the trifluoromethylated alkyne as cross-coupling product. Furthermore, the results suggest that oxygen is involved as oxidizing agent, and, apparently, the heteroleptic cuprates did not originate from the homoleptic ones, but presumably from neutral precursor species that cannot be observed by ESI mass spectrometry. ESI mass spectrometric experiments also demonstrated that the addition of organic iodides RI to solutions containing methyl argentate(I) complexes resulted in the formation of argentate(III) complexes. Upon collisional activation in the gas-phase, the latter species underwent reductive eliminations in which they preferentially released the cross-coupling product RMe. These findings point to the crucial role of argentates(I) and (III) in silver-mediated cross-coupling reactions. Moreover, the observed gas-phase reactivities significantly differ from those of previously studied organocpurates(III). Thus, the use of argentates may provide a future alternative to the popular organocuprates in synthesis.

In this study, electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to investigate selective recognition of G-quadruplex in the vascular endothelial growth factor (VEGF) gene with 12 small-molecule natural products. We found that kaempferol, a natural flavonol, shows the highest binding affinity among the 12 natural molecules. The results from ESI-MS and CD spectra indicated that kaempferol could enhance the thermal stability of the VEGF–G-quadruplex and showed selective recognition for the G-quadruplex in a solution consisting of the G-quadruplex and the corresponding duplex DNA.