ESI Mass Spectrometric Exploration of Selective Recognition of G-Quadruplex in c-myb Oncogene Promoter Using a Novel Flexible Cyclic Polyamide

In this research, electrospray ionization mass spectrometry (ESI-MS) was used to probe the binding selectivity of a flexible cyclic polyamide (cβ) to G-quadruplexes from the long G-rich sequences in the c-myb oncogene promoter. The results show that three G-rich sequences, including d[(GGA)₃GGTCAC(GGA)₄], d[(GGA)₄GAA(GGA)₄], and d[(GGA)₃GGTCAC(GGA)₄GAA(GGA)₄] species in the c-myb promoter can form parallel G-quadruplexes, and cβ selectively binds towards these G-quadruplexes over both several other G-quadruplexes and the duplex DNA. These properties of cβ have profound implications on future studies of the regulation of c-myb oncogene expression.

61<Superscript>ST</Superscript> ASMS Conference on Mass Spectrometry and Allied Topics

G-quadruplex nucleic acids can bind ammonium ions in solution, and these complexes can be detected by electrospray mass spectrometry (ESI-MS). However, because ammonium ions are volatile, the extent to which ESI-MS quantitatively could provide an accurate reflection of such solution-phase equilibria is unclear. Here we studied five G-quadruplexes having known solution-phase structure and ammonium ion binding constants: the bimolecular G-quadruplexes (dG₄T₄G₄)₂, (dG₄T₃G₄)₂, and (dG₃T₄G₄)₂, and the intramolecular G-quadruplexes dG₄(T₄G₄)₃ and dG₂T₂G₂TGTG₂T₂G₂ (thrombin binding aptamer). We found that not all mass spectrometers are equally suited to reflect the solution phase species. Ion activation can occur in the electrospray source, or in a high-pressure traveling wave ion mobility cell. When the softest instrumental conditions are used, ammonium ions bound between G-quartets, but also additional ammonium ions bound at specific sites outside the external G-quartets, can be observed. However, even specifically bound ammonium ions are in some instances too labile to be fully retained in the gas phase structures, and although the ammonium ion distribution observed by ESI-MS shows biases at specific stoichiometries, the relative abundances in solution are not always faithfully reflected. Ion mobility spectrometry results show that all inter-quartet ammonium ions are necessary to preserve the G-quadruplex fold in the gas phase. Ion mobility experiments, therefore, help assign the number of inner ammonium ions in the solution phase structure.      

Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Effects of Cations on Protein and Peptide Charging in Electrospray Ionization from Aqueous Solutions

The effects of eight different cations with ionic radii between 69 and 337 pm on the charging of peptides and proteins with electrospray ionization from aqueous acetate salt solutions are reported. Significant adduction occurs for all cations except NH₄ ⁺, and the average protein charge is lower when formed from solutions containing salts compared with solutions without salts added. Circular dichroism and ion mobility results show the protein conformations are different in pure water compared with salt solutions, which likely affects the extent of charging. The average charge of protein and peptide ions formed from solutions with Li⁺ and Cs⁺, which have Gibbs solvation free energies (GSFEs) that differ by 225 kJ/mol, is similar. Lower charge states are typically formed from solutions with tetramethylammonium and tetraethylammonium that have lower GSFE values. Loss of the larger cations that have the lowest GSFEs is facile when adducted protein ions are collisionally activated, resulting in the formation of lower analyte charge states. This reaction pathway provides a route to produce abundant singly protonated protein ions under native mass spectrometry conditions. The average protein and peptide charge with NH₄ ⁺ is nearly the same as that with Rb⁺ and K⁺, cations with similar GSFE and ionic radii. This indicates that proton transfer from NH₄ ⁺ to proteins plays an insignificant role in the extent of protein charging in native mass spectrometry.

On-Plate Self-Desalting and Matrix-Free LDI MS Analysis of Peptides With a Surface Patterned Sample Support

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH₄HCO₃ (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

Polythiophene-coated Fe3O4 nanoparticles as a selective adsorbent for magnetic solid-phase extraction of silver(I), gold(III), copper(II) and palladium(II)

We have developed a fast method for sensitive extraction and determination of the metal ions silver(I), gold(III), copper(II) and palladium(II). Fe₃O₄ magnetic nanoparticles were modified with polythiophene and used for extraction the metal ions without a chelating agent. Following extraction, the ions were determined by flow injection inductively coupled plasma optical emission spectrometry. The influence of sample pH, type and volume of eluent, amount of adsorbent, sample volume and time of adsorption and desorption were optimized. Under the optimum conditions, the calibration plots are linear in the 0.75 to 100 μg L^?1 concentration range (R²?>?0.998), limits of detection in the range from 0.2 to 2.0 μg L^?1, and enhancement factors in the range from 70 to 129. Precisions, expressed as relative standard deviations, are lower than 4.2 %. The applicability of the method was demonstrated by the successful analysis of tap water, mineral water, and river water.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

Fluorine Bonding Enhances the Energetics of Protein-Lipid Binding in the Gas Phase

This paper reports on the first experimental study of the energies of noncovalent fluorine bonding in a protein-ligand complex in the absence of solvent. Arrhenius parameters were measured for the dissociation of gaseous deprotonated ions of complexes of bovine β-lactoglobulin (Lg), a model lipid-binding protein, and four fluorinated analogs of stearic acid (SA), which contained (X =) 13, 15, 17, or 21 fluorine atoms. In all cases, the activation energies (E_a) measured for the loss of neutral XF-SA from the (Lg + XF-SA)^7– ions are larger than for SA. From the kinetic data, the average contribution of each?>?CF₂ group to E_a was found to be ~1.1 kcal mol^–1, which is larger than the ~0.8 kcal mol^–1 value reported for?>?CH₂ groups. Based on these results, it is proposed that fluorocarbon–protein interactions are inherently stronger (enthalpically) than the corresponding hydrocarbon interactions.

Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β-D-Galp-(1→3)-β-D-GalpNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Galp-(1→4)-β-D-Glcp (GM1os) to the cholera toxin B subunit homopentamer (CTB₅) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB₅ at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB₅, was found to be (3.2 ± 0.2) × 10⁶ M^–1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 10⁶ M^–1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

Thermally treated bare gold nanoparticles for colorimetric sensing of copper ions

We demonstrate a sensitive and rapid colorimetric assay for selective detection of copper ions based on the strong coordination between Cu(II) ions and the tetrahydroxyaurate anions [Au(OH)₄]^? on the surface of thermally treated bare gold nanoparticles (GNPs). The method for making the unmodified GNPs is simple and results in a nanomaterial with a highly specific response to Cu(II). The thermal treatment of the bare GNPs and the recognition of Cu(II) ions is accomplished in a single step within 5 min. The presence of Cu(II) causes the color to change from red to purple-blue. The limit of detection (LOD) is 0.04 μM of Cu(II) when using UV–vis spectrometry and ratioing the absorbances at 650 and 515 nm, respectively. The method also is amenable to bare eye (visual) inspection and in this case has an LOD of 2.0 μM of Cu(II).

Iodide-induced adsorption of lead(II) ion on a glassy carbon electrode modified with ferroferric oxide nanoparticles

Spherical Fe₃O₄ nanoparticles (NPs) were prepared by hydrothermal synthesis and characterized by scanning electron microscopy and X-ray diffraction. A glassy carbon electrode was modified with such NPs to result in a sensor for Pb(II) that is based on the strong inducing adsorption ability of iodide. The electrode gives a pair of well-defined redox peaks for Pb(II) in pH 5.0 buffer containing 10 mM concentrations of potassium iodide, with anodic and cathodic peak potentials at ?487 mV and ?622 mV (vs. Ag/AgCl), respectively. The amperometric response to Pb(II) is linear in the range from 0.10 to 44 nM, and the detection limit is 40 pM at an SNR of 3. The sensor exhibits high selectivity and reproducibility.

Soft Supercharging of Biomolecular Ions in Electrospray Ionization Mass Spectrometry

The charge states of biomolecular ions in ESI-MS can be significantly increased by the addition of low-vapor supercharging (SC) reagents into the spraying solution. Despite the considerable interest from the community, the mechanistic aspects of SC are not well understood and are hotly debated. Arguments that denaturation accounts for the increased charging observed in proteins sprayed from aqueous solutions containing SC reagent have been published widely, but often with incomplete or ambiguous supporting data. In this work, we explored ESI MS charging and SC behavior of several biopolymers including proteins and DNA oligonucleotides. Analytes were ionized from 100 mM ammonium acetate (NH₄Ac) aqueous buffer in both positive (ESI+) and negative (ESI–) ion modes. SC was induced either with m-NBA or by the elevated temperature of ESI capillary. For all the analytes studied we, found striking differences in the ESI MS response to these two modes of activation. The data suggest that activation with m-NBA results in more extensive analyte charging with lower degree of denaturation. When working solution with m-NBA was analyzed at elevated temperatures, the SC effect from m-NBA was neutralized. Instead, the net SC effect was similar to the SC effect achieved by thermal activation only. Overall, our observations indicate that SC reagents enhance ESI charging of biomolecules via distinctly different mechanism compared with the traditional approaches based on analyte denaturation. Instead, the data support the hypothesis that the SC phenomenon involves a direct interaction between a biopolymer and SC reagent occurring in evaporating ESI droplets.

Voltammetric and amperometric determination of hydrogen peroxide using a carbon-ceramic electrode modified with a nanohybrid composite made from single-walled carbon nanotubes and silver nanoparticles

A nanohybrid composite material was prepared from single-walled carbon nanotubes and silver nanoparticles, and used to fabricate a modified carbon-ceramic electrode. The preparation of the composite is facile and efficient. The nanohybrid composite deposited on the carbon-ceramic electrode was characterized by X-ray diffraction and cyclic voltammetry. The new electrode displays favorable electrocatalytic ability towards hydrogen peroxide (H₂O₂) and can be used to electrocatalytically reduce this species. Under the optimum conditions, the current measured during hydrodynamic amperometry is linearly related to the concentration of H₂O₂ over the concentration range from 0.01 to 8 mM, with a detection limit of 2?×?10^?7 M at a signal-to-noise ratio of 3 and sensitivity of 3.23 μA/mM. The electrode exhibits good reproducibility, long-term stability and negligible interference by dopamine, uric acid, and other important biological compounds. The electrode was successfully applied to the determination of H₂O₂ in honey samples, and the recovery was 101.2%.

Electron Transfer Dissociation of Photolabeled Peptides. Backbone Cleavages Compete with Diazirine Ring Rearrangements

Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK? + ?2H)²⁺ and (GL*GGK-NH₂?+?H)⁺ ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK? + ?2H)²⁺, (GL*GGK-NH₂?+?2H)²⁺,(GL*GGKK?+?2H)²⁺, (GL*GLK?+?2H)²⁺, and (GL*LGK?+?2H)²⁺ showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the diazirine ring. These side-chain dissociations resulted in eliminations of N₂H₃, N₂H₄, [N₂H₅], and [NH₄O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The diazirine moiety in GL*GGK-NH₂was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.

Nonenzymatic hydrogen peroxide sensor based on a glassy carbon electrode modified with electrospun PdO-NiO composite nanofibers

A glassy carbon electrode was modified with PdO-NiO composite nanofibers (PdO-NiO-NFs) and applied to the electrocatalytic reduction of hydrogen peroxide (H₂O₂). The PdO-NiO-NFs were synthesized by electrospinning and subsequent thermal treatment, and then characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. Factors such as the composition and fraction of nanofibers, and of the applied potential were also studied. The sensor exhibits high sensitivity for H₂O₂ (583.43 μA?·?mM^?1?·?cm^?2), a wide linear range (from 5.0 μM to 19 mM), a low detection limit (2.94 μM at an SNR of 3), good long term stability, and is resistant to fouling.

Nucleophilic Addition of Nitrogen to Aryl Cations: Mimicking Titan Chemistry

The reactivity of aryl cations toward molecular nitrogen is studied systematically in an ion trap mass spectrometer at 10² Pascal of nitrogen, the pressure of the Titan main haze layer. Nucleophilic addition of dinitrogen occurs and the nature of aryl group has a significant influence on the reactivity, through inductive effects and by changing the ground state spin multiplicity. The products of nitrogen activation, aryldiazonium ions, react with typical nitriles, aromatic amines, and alkynes (compounds that are relevant as possible Titan atmosphere constituents) to form covalently bonded heterocyclic products. Theoretical calculations at the level [DFT(B3LYP)/6-311++G(d,p)] indicate that the N₂ addition reaction is exothermic for the singlet aryl cations but endothermic for their triplet spin isomers. The –OH and –NH₂ substituted aryl ions are calculated to have triplet ground states, which is consistent with their decreased nitrogen addition reactivity. The energy needed for the generation of the aryl cations from their protonated precursors (ca. 340 kJ/mol starting with protonated aniline) is far less than that required to directly activate the nitrogen triple bond (the lowest energy excited state of N₂ lies ca. 600 kJ/mol above the ground state). The formation of aza-aromatics via arene ionization and subsequent reactions provide a conceivable route to the genesis of nitrogen-containing organic molecules in the interstellar medium and Titan haze layers.

Acetone sensor based on solvothermally prepared ZnO doped with Co3O4 nanorods

This paper describes a reliable and sensitive method for sensing dissolved acetone using doped nanomaterials. Large-scale synthesis of ZnO nanorods (NRs) doped with Co₃O₄ was accomplished by a solvothermal method at low temperature. The doped NRs were characterized in terms of their morphological, structural, and optical properties by using field-emission scanning electron microscopy coupled with energy-dispersive system, UV-Vis., Fourier transform IR, X-ray diffraction, and Xray photoelectron spectroscopy. The calcinated (at 400 °C) doped NRs are shown to be an attractive semiconductor nanomaterial for detecting acetone in aqueous solution using silver electrodes. The sensor exhibits excellent sensitivity, stability and reproducibility. The calibration plot is linear over a large concentration range (66.8 μM to 0.133 mM), displays high sensitivity (~3.58 μA cm^?2 mM^?1) and a low detection limit (~14.7?±?0.2 μM; at SNR of 3).

The interaction of platinum-based drugs with native biologically relevant proteins

This study focuses on the identification of the products that are formed upon binding of therapeutically relevant platinum complexes to proteins like β-lactoglobulin A (LGA), human serum albumin (HSA), or human hemoglobin (HB). The respective proteins were incubated with the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin. LGA was selected as the model protein in addition to the two most abundant blood proteins HSA and HB. In case of the model protein, the effect of free thiol groups on the affinity of cisplatin, carboplatin, and oxaliplatin was investigated by means of liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS). The reduced form of LGA, which contains four free thiol groups more than the native LGA, shows a much higher affinity to the platinum-based drugs. By means of liquid chromatography coupled to inductively coupled plasma mass spectrometry, the reaction behavior of the platinum-based drugs towards HSA and HB was investigated under different conditions considering the chloride concentration (4 or 100 mM) and the incubation time (24 and 48 h). In case of carboplatin, less than 6 % protein-bound platinum was detected. However, both cisplatin and oxaliplatin display a high affinity to the proteins investigated. Further information was obtained by means of LC/ESI-ToF-MS. In case of oxaliplatin, the complex [Pt(DACH)]²⁺ (DACH?=?C₆N₂H₁₄) was identified interacting with HSA and HB. For cisplatin, different results were observed for the two proteins. The complex [Pt(NH₃)₂Cl]⁺ interacted predominantly with HSA and [Pt(NH₃)₂]²⁺ with HB.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m₂ and m₂+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

Solid phase extraction of Cd(II) and Pb(II) using a magnetic metal-organic framework, and their determination by FAAS

We describe a novel magnetic metal-organic framework (MOF) for the preconcentration of Cd(II) and Pb(II) ions. The MOF was prepared from the Fe₃O₄-pyridine conjugate and the copper(II) complex of trimesic acid. The MOF was characterized by IR spectroscopy, elemental analysis, SEM and XRD. A Box-Behnken design through response surface methodology and experimental design was used to identify the optimal parameters for preconcentration. Extraction time, amount of magnetic MOF and pH value were found to be critical factors for uptake, while type, volume, concentration of eluent, and elution time are critical in the elution step. The ions were then determined by FAAS. The limits of detection are 0.2 and 1.1 μg?L^?1 for Cd(II), and Pb(II) ions, respectively, relative standard deviations are <4.5% (for five replicates at 50 μg?L^?1 of Cd(II) and Pb(II) ions), and the enrichment capacity of the MOF is at around 190 mg?g^?1 for both ions which is higher than the conventional Fe3O4-pyridine material. The magnetic MOF was successfully applied to the rapid extraction of trace quantities of Cd(II) and Pb(II) ions in fish, sediment, and water samples.