Electrospray Ionization Mass Spectrometry Measurements Research Articles

Use of Fluorescent DNA-Templated Gold/Silver Nanoclusters for the Detection of Sulfide Ions

We have developed a one-pot approach to prepare fluorescent DNA-templated gold/silver nanoclusters (DNA-Au/Ag NCs) from Au(3+), Ag(+), and DNA (5'-CCCTTAATCCCC-3') in the presence of NaBH(4) in order to detect sulfide (S(2-)) ions on the basis of fluorescence quenching. The as-prepared DNA-Au/Ag NCs have been characterized by UV-vis absorption, fluorescence, circular dichroism, X-ray photoelectron spectroscopy, and electrospray ionization-mass spectrometry measurements. Relative to DNA-Ag NCs, DNA-Au/Ag NCs are much more stable in high ionic strength media (e.g., 200 mM NaCl). The quantum yield of the as-prepared DNA-Au/Ag NCs is 4.5%. We have demonstrated that the fluorescence of DNA-Au/Ag NCs is quenched by S(2-) ions through the interaction between sulfide ions and gold/silver atoms/ions, a result which leads to changes in the conformation of the templated DNA from packed hairpin to random coil structures. These changes in fluorescence intensity allow sensitive detection of S(2-) ions at concentrations as low as 0.83 nM. To minimize interference from I(-) ions for the detection of S(2-) ions using the DNA-Au/Ag NCs, the addition of sodium peroxydisulfate to the solution is essential. We have validated the practicality of this probe for the detection of S(2-) ions in hot spring and seawater samples, demonstrating its advantages of simplicity, sensitivity, selectivity, and low cost.

Binding of Clostridium difficile toxins to human milk oligosaccharides

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3)M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.

Identification of Active Sites of Biomolecules II: Saccharide and Transition Metal Ion in Aqueous Solution

We discuss the coordination mechanism of Fe(III) and methyl-alpha-mannopyranoside in aqueous solution using a recently presented integrated approach comprising ab initio electronic structure calculations, molecular dynamics simulations, and mass spectrometric measurements. First principles Car-Parrinello molecular dynamics (CPMD) simulations find that a single Fe(III) ion interacts with specific hydroxyl groups of the saccharide in aqueous solution. Specifically, we find that one Fe(III) ion complexed to methyl-alpha-mannopyranoside also associates with two water molecules. These simulations are in accord with electrospray ionization mass spectrometry measurements involving guided ion beam hydration measurements, which reveal an optimal coordination number of four about the Fe(III) ion. CPMD simulations identified specific intramolecular and intermolecular hydrogen bonding interactions that have an impact on the conformation of the saccharide and on the coordination with Fe(III); in contrast, classical molecular dynamics simulations were insensitive to these effects. This study illustrates the strength of ab initio molecular dynamics simulations, chemical reactivity calculations, and natural partial charge analysis coupled with mass spectrometric measurements in identifying the active sites of biomolecules toward ligands and for studying the complexation and coordination chemistry associated with substrate and ligand interactions relevant to the design of biochemical syntheses, drugs, and biomarkers in medicine.

Investigation of non-covalent complexes of glutathione with common amino acids by electrospray ionization mass spectrometry

To study the non-covalent interaction between glutathione and common amino acids. A stoichiometry of glutathione and common amino acids were mixed to reach the equilibrium, and then the mixed solution was investigated by electrospray ionization mass spectrometry (ESI-MS). The binding of the complexes was further examined by collision-induced dissociation (CID) in a tandem mass spectrometer as well as UV spectroscopy. To avoid distinct ionization efficiency discrepancy and signal suppression in the ESI-MS measurements, the interaction between glutathione (GSH) and glutamate (Glu) was quantitatively evaluated. The total concentrations and series of m/z of peak intensities for glutathione and amino acids could be achieved, respectively. Due to the existence of some oligomeric species arising from glutathione or amino acids, an improved calculation formula was proposed to calculate the dissociation constants of glutathione binding to amino acids. The ESI mass spectra revealed that glutathione could interact easily with Met, Phe, Tyr, Ser, or Ile to form non-covalent complexes. The binding of the complexes was further confirmed by CID experiments in a tandem mass spectrometer as well as UV spectroscopy. Moreover, an improved calculation formula was successfully applied to determine the dissociation constants of glutathione binding to Glu, His, or Gln. Finally, a possible formation mechanism for the complexes of glutathione with amino acids was proposed. The reduced polypeptide gamma-glutathione can interact with each of 8 common amino acids, including Glu, His, and Gln to form non-covalent complexes with different affinity.

Probing Non-Covalent Complexes of Glutathione with D-Amino Acids by Mass Spectrometry

To investigate the non-covalent interaction between glutathione and D-amino acids, stoichiometric reduced glutathione (GSH) and three D-amino acids, including D-phenylalanine (D-Phe), D-histidine (D-His), and D-glutamine (D-Gln), were mixed respectively, and then incubated at room temperature for 1 h to reach equilibrium. The electrospray ionization mass spectrometry (ESI-MS) results indicated that glutathione and three D-amino acids could form non-covalent complexes in physiological pH conditions, respectively. The binding of glutathione to D-amino acid was further confirmed by collision-induced dissociation (CID) in a tandem mass spectrometer. Additionally, the complexes exhibited different features and properties from the reactants in UV spectroscopy, which also confirmed the results of ESI-MS. To avoid distinct ionization efficiency discrepancy and signal suppression in ESI-MS measurements, the interaction between glutathione (GSH) and D-glutamine (Gln) was quantitatively evaluated. A series of samples with different initial concentrations of glutathione and D-amino acids were mixed, and then series of peak intensities for different species in the mixture were achieved by ESI-MS. The dissociation constants of three complexes formed by glutathione and D-amino acids were calculated. The calculation results revealed that the reduced tripeptide γ-glutathione could interact with D-amino acids to form non-covalent complexes with different affinities, the stabilities of the three complexes increased gradually according to the order of D-glutamine, D-phenylalanine, and D-histidine.

Anionic CunIn Cluster-Based Architectures Induced by In Situ Generated N-Alkylated Cationic Triazolium Salts

Five new copper(I) iodide coordination polymers, (Cu10I10)(Cu6I6)[(Cu(Bta)2]3−·(DMBta)4+·(I−)·x(I2) (x ≈ 1.5) (1), [Cu2I2(MBta)] (2), [Cu2I2(MTa)] (3), (Cu5I5)[Cu(Ta)2]−·(DETa)+ (4), and [Cu3I(Bta)2] (5) (Bta = benzotriazole, DMBta = 1,3-N-dimethylbenzotriazolium, MBta = 1-N-methylbenzotriazole, MTa = 1-N-methyltriazole, Ta = 1,2,3-triazole, DETa = 1,3-N-diethyltriazolium), have been prepared by the solvo(hydro)thermal reactions of CuI, KI, and benzotriazole (or 1,2,3-triazole) in methanol (or ethanol or water) and structurally characterized. Complex 1 shows a highly unusual two-dimensional (2D) → three-dimensional (3D) interpenetrated anionic network containing two new discrete neutral Cu10I10/Cu6I6 cluster units, and complex 4 consists of 2D anionic layers with Cu5I5 cluster units, both of which involve in situ di-N-alkylated cationic triazolium to induce the anionic frameworks. Complexes 2 and 3 both contain neutral in situ mono-N-alkylated ligands and exhibit similar one-dimensional (1D) double-stranded stairlike ribbon structures. Complex 5 exhibits a 2D simple sheet structure containing nonalkylated Bta− ligands. A mechanism of the solvo(hydro)thermal reaction is proposed. As evidenced by means of electrospray ionization mass spectrometry measurements, a simultaneous redox, N-alkylated and self-assembly reaction occurs.

Monovalent cation-promoted ordering of a glycine-rich cyclic peptide

This report describes an accelerated self-assembly of a synthetic cyclic hexapeptide in the presence of alkali metal ions. Time-dependent aggregation of hexapeptide was considerably influenced upon co-incubation with monovalent metal ions, of which K + afforded the most significant effect both on the time-scale required for self-assembly and on the morphology of aged structures. Metal ion adducts formation ability of the hexapeptide was confirmed by electrospray ionization mass spectrometry measurements and 13C NMR spectrometry. The effect of metal ion binding on peptide structure was also probed by circular dichroism, optical microscopy, and scanning electron microscopy. K + ions not only interacted more efficiently with the hexapeptide enabling it to reach conformational state(s) conducive for self-assembly, but also altered the morphologies of the aged peptide fibers, when compared to the unmetalated peptide.

Effect of the Electrolyte Concentration on Cation Binding in Aqueous Surfactant Solutions

Electrospray ionisation mass spectrometry (ESI-MS) is used for the qualitative and quantitative study of ion binding to interfaces. The ESI-MS measurements are carried out in aqueous solutions of sodium dodecyl sulphate (SDS) in the presence of NaCl, NH(4)ClO(4) , KCl, or Mg(ClO(4))(2) . The effect of the electrolyte concentration on the fractions, y(cation(+) ), of the DS(-) monomers bound to the cations is shown. Moreover, the binding stability of different cations with DS(-) monomers is determined versus the electrolyte concentration. This stability and the y(Na(+) ) values (the Na(+) ions derived from SDS), estimated in the presence of a given electrolyte, are quantitatively correlated with the electrolyte cation effect on the water structure. We also present calibration curves from which the molar concentrations of the sodium ions (derived from SDS) and of the other cations (derived from the electrolyte), bound to the SDS-micelle surface at a given electrolyte concentration, can be obtained. Besides, specific ion effects (Hofmeister effects) are considered for 1:1 electrolytes.

Synthesis, Crystal Structure, and Characterization of New Tetranuclear Ag(I) Complexes with Triazole Bridges

The self-assembly of Ag(I) ions with 3,5-dimethyl-4-amino-1,2,4-triazole (L1) and 4-salicylideneamino-1,2,4-triazole (L2) gave two novel complexes, [Ag4(mu2-L1)6][Ag4(mu2-L1)6(CH3CN)2](ClO4)8.2H2O (1) and [Ag4(mu2-L2)6(CH3CN)2](AsF6)4.2H2O (2), both of which contain tetranuclearic clusters constructed via Ag(I) ions and six N1,N2-bridged triazoles with a Ag4N12 core. When 4-(6-amino-2-pyridyl)-1,2,4-triazole (L3) was employed, {[Ag4(mu2-L3)4(mu3-L3)2](CF3SO3)4.H2O}n (3), {[Ag4(mu2-L3)4(mu3-L3)2](ClO4)4}n (4), and {[Ag4(mu2-L3)2(mu3-L3)4](PF6)4.CH3CN.0.75H2O}n (5) were isolated. 3 and 4 are 1D polymers, while 5 is a 2D polymer. 1D and 2D coordination polymers are constructed via the self-assembly of Ag4N12 cores as secondary building units (SBUs). The connection of these SBUs can be represented as a ladderlike structure for 1D polymers and a 4.8(2) net for 2D polymers. Electrospray ionization mass spectrometry measurements and NMR (1H and 13C) studies demonstrate that the tetranuclear SBU retains its integrity and the coordination polymers decompose into the tetranuclear Ag4N12 core in solution. 2 exhibits blue emission in the solid state and green emission in solution at ambient temperature. Strong blue fluorescence for complexes 3-5 in the solid state can be assigned to the intraligand fluorescent emission.

Electrospray mass spectrometry determination of poly(oxazolidine acetal).

The polycondensation of terephthalaldehyde with 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS), or of terephthaldehyde with 2,2'-(1,4-phenylene)-bis-1,3-(4,4-dihydroxymethyl)oxazolidine (bis-oxazolidine), using acidic catalysts, leads to the formation of linear polymers and macrocyclic compounds. The structure of the polymer was determined by (1)H-, (13)C-NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) analysis. ESI-MS measurements were used to study the details of the polymer structure and support the alternating nature of equimolar amounts of oxazolidine and the cyclic acetal of terephthaldehyde in these macromolecules.

Inhibition of 3C Protease from Human Rhinovirus Strain 1B by Peptidyl Bromomethylketonehydrazides

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in anEscherichia colistrain which also overexpressed the rareargUtRNA. The protease was isolated from inclusion bodies, refolded, and exhibited akcat/Km= 3280 M−1s−1using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease withkinact/Kinactvalues as high as 23,400 M−1s−1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to thekinact/Kinactrate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145–154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.

Purification and mass spectrometry-assisted sequencing of basic antifungal proteins from seeds of pumpkin ( Cucurbita maxima)

A number of different basic polypeptides with in vitro antifungal activity were isolated from the soluble and cell wall-derived fractions of seeds of pumpkin ( Cucurbita maxima) using batchwise chromatography on carboxymethyl-cellulose (CM52) and reverse-phase HPLC. Polypeptide masses were determined by electrospray ionisation mass spectrometry (ESMS). Reverse-phase HPLC, on a C8 column subjected to a gradient of increasing CH 3CN concentration in 0.1% TFA, yielded successive peaks 1–4 containing a multiplicity of components with the following masses (most abundant components underlined): peak 1 and peak 2 ( 4650 , 4779, 4850, 4890, 4961, 5097 and 5253 Da), peak 3 (5253, 7084, 7194 , 7308 and 7350 Da) and peak 4 (11 696 Da). An additional 2249 Da component was isolated from the cell-wall enriched fraction, but was not present in the soluble fraction. Fractions containing the major 2249, 4650 and 11 696 Da components exhibit antifungal activity against a variety of fungi. The 11 696 Da component is a napin-like protein composed of a 3700 Da small chain (CS1) and an 8004 Da large chain (CL1) that are joined by two of the four disulphide linkages in the oxidised complex. The complete amino acid sequences of the napin-like protein small and large chains, the 2249 Da cell-wall protein and the 4650 Da protein (and its associated homologues) were determined from Edman sequencing, previously published cDNA sequence data and corroborative ESMS measurements of the masses of tryptic fragments and/or intact proteins. Within experimental uncertainty, the calculated masses for the napin-like protein major small and large chains and for the corresponding oxidised complex are in exact agreement with their observed masses. Unlike many other basic antifungal proteins, the 11 696 Da napin-like protein is neither a substrate for Ca 2+-dependent protein kinase (CDPK) nor a calmodulin (CaM) antagonist. CS1 and CL1 do not have the putative `two-sided' α-helical structural element found in all the related napin and napin-like protein small and large chains shown to be CaM antagonists.

HIV-1 Tat Peptide Binding to TAR RNA by Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent complexes formed from the interaction between HIV-1 Tat peptide and Tat protein with TAR RNA. Both positive ion and negative ion ESI mass spectra showed a maximum stoichiometry of 3:1 between Tat peptide and TAR RNA. However, the higher order complexes only occurred at high relative concentrations of Tat peptide. The 1:1 Tat peptide-TAR RNA complex is believed to involve only specific interactions, whereas the higher order complexes involve nonspecific interactions. Relative binding affinities between Tat peptide and TAR RNA and its various mutants (TAR missing the three-nucleotide bulge, TAR with a poly(ethylene glycol) linker in the bulge region, and TAR with a poly(ethylene glycol) linker in the loop region) can be differentiated by competitive binding experiments and ESI-MS measurements. The gas phase mass spectrometry experiments are consistent with solution phase studies, as they show that mutations in the bulge region reduce TAR RNA affinity to Tat peptide.

Electrospray Ionization Mass Spectrometry: Part II: Applications in Characterization of Peptides and Proteins

In part I of this article, we discussed the theory and operational features of electrospray ionization mass spectrometry (ESI-MS). Herein, we survey a few common applications of ESI-MS. The symbiotic relationship between biological chemists and mass spectrometrists has lead to rapid development of commercial ESI-MS systems and their utilization by an ever growing number of researchers. It is our hope in this article to answer the question: What do ESI-MS measurements tell researchers and how does this information assist their research?