Identification of bismuth-thiolate-carboxylate clusters by electrospray ionization mass spectrometry

The known thermal and hydrolytic stability of bismuth-sulfur bonds indicates that biological targets for bismuth likely involve thiol or thiolate functionalities, such as in L-cysteine. Complexes of bismuth with cysteine or other thiol-carboxylic acid ligands have been isolated and characterized providing a preliminary view of the potential participation of these functional groups in the biochemical mechanisms involving bismuth. A broader assessment of bismuth-thiolate interactions has been possible using electrospray ionization mass spectrometry (ESI-MS). A wide range of complexes has been observed containing mercaptosuccinic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, and/or 2-amino-3-mercaptopropionic acid (cysteine). The identification of various multibismuth multiligand cluster ions defines new chemistry for bismuth.     

False positives and the detection of cyclodextrin inclusion complexes by electrospray mass spectrometry

The results of previous works that have claimed to detect cyclodextrin inclusion complexes via the “soft” ionization technique of electrospray ionization mass spectrometry are revisited. A more extensive study of cyclodextrin mixtures with amino acids and small peptides demonstrates that amino acid and peptide “complexes” are detected by electrospray mass spectrometry regardless of the presence (or not) of an aromatic moiety on the side chain. Amino acids that may be least likely to form hydrophobic inclusion complexes with cyclodextrin in solution generally show the most intense complex ions. The data suggest that these “complexes” are, in all likelihood, electrostatic adducts formed during the electrospray process. Systematic controls are suggested to ensure that “false positives” do not negate many of the claims concerning the detection of solution-derived noncovalent compounds.     

Amino acid cluster formation studied by electrospray ionization mass spectrometry

The association properties of natural and non-natural amino acids were studied in detail using electrospray ionization mass spectrometry. The results show a highly diverse cluster formation behavior of amino acids. There are differences regarding the degree of clustering (average cluster size), the presence or absence of one or several 'magic' clusters of special stability and the influence of chirality on cluster stability. Cluster formation does not show a good correlation with simple physico-chemical properties (such as solubility), indicating that it is a specific process and not only a simple aggregation during evaporation/ionization. A systematic study of cluster formation of serine derivatives reveals that all functional groups play a prominent role in the binding of the magic octamer. The results support the idea of the zwitterionic character of the octamer. Electrospray ionization of the side-chain acetylated serine shows the formation of a very stable tetramer with a strong preference for homochirality. The results suggest that Ser8 is made up of two tetramer subunits, held together by hydrogen bonds of the side-chain.     

Determination of dissociation constants of cyclodextrin-ligand inclusion complexes by electrospray ionization mass spectrometry

The inclusion complexes of four ligands binding to cyclodextrins (CDs) were studied by electrospray ionization mass spectrometry (ESI-MS) and the dissociation constants of the complexes were obtained. The 1:1 stoichiometric inclusion complex was found in the system of CD and fenbufen or aspirin. The obtained KD values of the inclusion complexes of fenbufen binding to alpha-CD and to beta-CD are 4.38x10(-4) mol L(-1) and 2.12x10(-4) mol L(-1), respectively. The KD values of the inclusion complexes of alpha-CD-aspirin and beta-CD-aspirin are 3.33x10(-4) mol L(-1) and 1.83x10(-4) mol L(-1), respectively. A non-linear least squares regression method was applied to validate the results which were consistent with each other. For the system of tetracycline hydrochloride and CD, the 1:1 and 1:2 stoichiometric inclusion complexes were found in the mass spectra. The KD,1 and KD,2 values of the 1:1 and 1:2 stoichiometric inclusion complexes of alpha-CD and tetracycline hydrochloride are 4.47x10(-4) mol L(-1) and 6.51x10(-4) mol L(-1), respectively, and those of beta-CD and tetracycline hydrochloride are 2.26x10(-4) mol L(-1) and 8.57x10(-4) mol L(-1), respectively. For the system of norfloxacin and CD, besides the 1:1 and 1:2 inclusion complexes, the 1:3 stoichiometric inclusion complex was also found. The KD,1, KD,2 and KD,3 of alpha-CD and norfloxacin inclusion complexes are 4.61x10(-4) mol L(-1), 6.05x10(-4) mol L(-1) and 1.45x10(-3) mol L(-1), respectively. The three KD values of beta-CD and norfloxacin are 1.96x10(-4) mol L(-1), 4.93x10(-4) mol L(-1) and 1.15x10(-3) mol L(-1), respectively.     

G-quadruplex formation of thrombin-binding aptamer detected by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry detected the formation of the G-quadruplex structure of the thrombin-binding aptamer, d(GGTTGGTGTGGTTGG), and established its specific interaction with metal ions. One piece of evidence that the bonding in the gas phase is via the G-quadruplex form is the enhanced binding, with respect to other metal ions, of the aptamer with Sr2+, Pb2+, Ba2+, and K+, which are of similar size. Another is the lack of specific binding with controls in which the G's are replaced with A's. The most convincing evidence is the extent of H/D exchange of the gas-phase aptamer as compared to that bound to K+ and Sr2+. The latter two complexes exchange six and nine fewer H's, indicating a significant increase in protection upon binding to the metals. Mass spectrometry will be an important tool in understanding G-quadruplexes, which are particularly important in DNA telomers.     

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.     

Identification of self-assembly products from N-phosphoamino acids by electrospray ionization mass spectrometry

The self-assembly products of N-(O,O'-diisopropyl)-L-alpha-amino acids in aqueous media, N-(O,O'-diisopropyl)phosphoryl dipeptides and tripeptides, were identified by electrospray ionization mass spectrometry. A stepwise fragmentation of the [M + H](+) ions from the C-terminus of N-phosphopeptides was observed. Copyright 2000 John Wiley & Sons, Ltd.     

Measurement of inclusion complex formation between cyclophane and biological relevant amino acids using electrospray ionization, cold-spray ionization and fast atom bombardment mass spectrometry

The investigation of the host-guest complex formations between cyclophane (TGDMAP) (1) as a host and L-acidic amino acids such as L-glutamic acid (Glu) and L-aspartic acid (Asp) as guests was carried out using fast atom bombardment (FAB), electrospray ionization (ESI) and cold-spray ionization (CSI) mass spectrometry (MS). The stability constant (K(s)) values obtained by the three different MS methods almost agreed. However, the complex ion peaks of a novel cyclophane (CPCn) (2) with Glu and Asp were not observed in FAB-MS. Then, these host-guest complex formations by use of CSI-MS and ESI-MS was examined, as the results, these complex ion peaks were observed clearly and the measurement values by the two MS methods are mostly in agreement. It was concluded that ESI-MS and CSI-MS are available for the determination of K(s) value as well as FAB-MS.     

Characterization of the rutin-metal complex by electrospray ionization tandem mass spectrometry.

According to the strong application background of bioflavonoid and metal-flavonoid complexes, novel electrospray ionization tandem mass spectrometry (ESI-MSn) was applied to investigate the structure and fragmentation mechanism of transition metal-rutin complexes. In the full-scan mass spectra, different stoichiometric ratios of rutin-metal complexes were found. In the reaction between rutin and Cu, four kinds of complexes with four different stoichiometric ratios were produced. In the reaction between rutin and Zn, Mn(II), and Fe(II), only two kind of complexes with stoichiometric ratios of 1:1 and 1:2 occured. In further tandem mass spectrometric experiments of different rutin-metal complexes, product fragments came from the neutral loss of the external rhamnose and the internal glucose unit, oligosaccharide chain, aglycone, and small organic molecules. According to the MSn data, we proposed a mechanism for all fragments of the rutin-Cu complex A and the structure of two rutin-Cu complexes, C and D.     

Identification of ethyl pyruvate and dihydrocinchonidine adducts by electrospray ionization mass spectrometry

Several ethyl pyruvate and dihydrocinchonidine adducts, formed by non-covalent interactions with alkali cations, have been identified for the first time using electrospray ionization mass and tandem mass spectrometry. This type of adduct may have an important role in asymmetric reactions of pyruvates in the presence of cinchonas.     

Efficient determination and evaluation of model cyclodextrin complex binding constants by electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for the detection of cyclodextrin noncovalent complexes, complementing previously established methods. Host-guest complexes formed in solution are also stable for characterization by ESI in the gas phase. This paper reports the first investigations to characterize the stability of three inclusion complexes between beta-cyclodextrin (beta-CD) and three model "guest" molecules, by determining the cyclodextrin compound complex stability constant (K(st)) with the use of mass spectrometric studies. The relative signal intensity of the complexes were monitored in the positive ion mode by mixing each "guest" molecule with an up to 50-fold molar excess of betaCD. A novel linear equation, similar to Benesi-Hildebrand, was derived allowing the determination of K(st) for 1:1 stoichiometry in all complexes. These values were compared with the K(st) obtained by spectrophotometric experiments and they were evaluated to be slightly different, indicating the validity of the described method.     

Characterization of a cross-linked DNA-Endonuclease VIII repair complex by electrospray ionization mass spectrometry

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.     

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.     

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.     

电喷雾质谱在金属配合物研究中的应用

综述了电喷雾质谱(ESI-MS)在金属配合物分析中的应用,详细介绍了电喷雾质谱在测定金属配合物的分子量、表征金属配合物的结构、分析金属离子与配体的相互作用及相关机理等方面的研究进展.     

New determination methods of halides and cyanide ions by electrospray ionization mass spectrometry based on ternary complex formation

Determination methods of halide ions (X(-) = F(-), Cl(-), Br(-) and I(-)) by electrospray ionization mass spectrometry (ESIMS) were developed, where negative ions of the ternary complexes of group-13 elements, nitrilotriacetic acid (NTA), and halides were measured. In particular, these halides were simultaneously determined by measuring [InX(nta)](-), and the limits of detection (LODs) were 1.1 μmol dm(-3) for F(-), 0.32 μmol dm(-3) for Cl(-), 3.8 nmol dm(-3) for Br(-), and 1.6 nmol dm(-3) for I(-), respectively. This approach was extended to the determination of CN(-), where the ternary complex of Cu(II), CN(-) and 4-(2-pyridylazo)resorcinol (PAR), i.e., [(63)Cu(II)(CN)(par)](-) (m/z 302) was measured. The LOD for CN(-) was 20 nmol dm(-3).     

On the specificity of cyclodextrin complexes detected by electrospray mass spectrometry

Alpha-cyclodextrin complexes with linear alpha,omega-dicarboxylic acids were investigated by electrospray mass spectrometry. These hydrophobic complexes are known to have an equilibrium binding constant that increases with the diacid chain length. However, the electrospray mass spectrometry (ES-MS) spectra showed that the relative intensity of the complex did not vary significantly with chain length. This contradiction is caused by a contribution of nonspecific adducts to the signal of the complex in ES-MS. In order to estimate the contribution of nonspecific adducts to the total intensity of the complexes with alpha-cyclodextrin, the comparison was made between alpha-cyclodextrin and maltohexaose, the latter being incapable of making inclusion complexes in solution. The signal observed for complexes between diacids and maltohexaose can only result from nonspecific electrostatic aggregation, and is found to be more favorable with the shorter diacids. This is also supported by MS/MS experiments. A procedure is described which allows estimation of the contribution of the nonspecific complex in the spectra of the complexes with alpha-cyclodextrin by using the relative intensity of the complex with maltohexaose. The contribution of the specific complex to the total signal intensity is found to increase with the diacid chain length, which is in agreement with solution behavior.     

Intact skin analysis by desorption electrospray ionization mass spectrometry

In vivo skin analysis by Desorption Electrospray Ionization was characterized on healthy human volunteers by directing pneumatically assisted electrospray directly onto their fingertips. In order to eliminate the risk of electric shock, a high ohmic resistor was built into the system. Positive ion DESI-MS analysis yields low intensity spectra, while negative ion spectra feature a number of various biogenic carboxylic acids. Compounds of external origin and excreted molecules were found to have different analysis kinetics, with the exception of highly hydrophobic species. The difference was demonstrated in the case of nicotine and cotinine. Pharmacokinetic studies were performed using a rat animal model. The kinetics of the anesthetic ketamine was followed by DESI, and results were in agreement with off-line HPLC-MS blood analysis. Using a similar approach for N,N'-dimethylthiourea (DMTU), a novel method was developed for the real-time quantification of oxidative stress. DMTU was administered to the animals, and the ratio of the molecule and its oxidized form was monitored from the skin surface. The ratio was found to be highly sensitive to experimentally induced diabetes mellitus type I and angiotensin-induced chronic oxidative stress. It was concluded that the method has a number of potential applications in the fields of forensics, pharmacology and clinical chemistry.     

High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry

A newly developed high-throughput desorption electrospray ionization (DESI) source was characterized in terms of its performance in quantitative analysis. A 96-sample array, containing pharmaceuticals in various matrices, was analyzed in a single run with a total analysis time of 3 min. These solution-phase samples were examined from a hydrophobic PTFE ink printed on glass. The quantitative accuracy, precision, and limit of detection (LOD) were characterized. Chemical background-free samples of propranolol (PRN) with PRN-d₇ as internal standard (IS) and carbamazepine (CBZ) with CBZ-d₁₀ as IS were examined. So were two other sample sets consisting of PRN/PRN-d₇ at varying concentration in a biological milieu of 10% urine or porcine brain total lipid extract, total lipid concentration 250 ng/μL. The background-free samples, examined in a total analysis time of 1. 5 s/sample, showed good quantitative accuracy and precision, with a relative error (RE) and relative standard deviation (RSD) generally less than 3% and 5%, respectively. The samples in urine and the lipid extract required a longer analysis time (2. 5 s/sample) and showed RSD values of around 10% for the samples in urine and 4% for the lipid extract samples and RE values of less than 3% for both sets. The LOD for PRN and CBZ when analyzed without chemical background was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol analyzed in 10% urine, and 200 fmol when analyzed in the brain lipid extract.     

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. This phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established.