A study of oxaliplatin–nucleobase interactions using ion trap electrospray mass spectrometry

Oxaliplatin is an important anti-cancer drug that has been approved for the treatment of colorectal cancer. It is known that oxaliplatin, like other Pt-based drugs, interacts with DNA to form cytotoxic Pt-DNA adducts that disrupt important biological processes such as DNA replication and protein synthesis. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of oxaliplatin with DNA nucleobases. It was shown that oxaliplatin formed adducts with all four DNA nucleobases when present individually and in combination in solution. Multiple-stage tandem mass spectrometry (MSⁿ) enabled the fragmentation pathways of each adduct to be established. In addition, proposed structures for each product ion were obtained from the MS data. When all four bases were present together with the drug at near-equal molar concentrations, adducts containing predominantly adenine and guanine were formed, confirming that the drug preferentially binds to these nucleobases. A large molar excess of drug was required to ensure the formation of cytosine and thymine adducts in the presence of adenine and guanine. Even with a large excess of oxaliplatin, only mono-adducts of these nucleobases were observed when all four nucleobases were present. Figure Schematic of a linear ion trap mass spectrometer being used to isolate the diadduct of guanine with oxaliplatin showing the characteristic isotope pattern due to ¹⁹⁴Pt, ¹⁹⁵Pt and ¹⁹⁶Pt.     

A systematic study on the influence of carbon on the behavior of hard-to-ionize elements in inductively coupled plasma–mass spectrometry

A systematic study on the influence of carbon on the signal of a large number of hard-to-ionize elements (i.e. B, Be, P, S, Zn, As, Se, Pd, Cd, Sb, I, Te, Os, Ir, Pt, Au, and Hg) in inductively coupled plasma–mass spectrometry has been carried out. To this end, carbon matrix effects have been evaluated considering different plasma parameters (i.e. nebulizer gas flow rate, r.f. power and sample uptake rate), sample introduction systems, concentration and type of carbon matrix (i.e. glycerol, citric acid, potassium citrate and ammonium carbonate) and type of mass spectrometer (i.e. quadrupole filter vs. double-focusing sector field mass spectrometer). Experimental results show that P, As, Se, Sb, Te, I, Au and Hg sensitivities are always higher for carbon-containing solutions than those obtained without carbon. The other hard-to-ionize elements (Be, B, S, Zn, Pd, Cd, Os, Ir and Pt) show no matrix effect, signal enhancement or signal suppression depending on the experimental conditions selected. The matrix effects caused by the presence of carbon are explained by changes in the plasma characteristics and the corresponding changes in ion distribution in the plasma (as reflected in the signal behavior plot, i.e. the signal intensity as a function of the nebulizer gas flow rate). However, the matrix effects for P, As, Se, Sb, Te, I, Au and Hg are also related to an increase in analyte ion population caused as a result of charge transfer reactions involving carbon-containing charged species in the plasma. The predominant specie is C⁺, but other species such as CO⁺, CO₂⁺, C₂⁺ and ArC⁺ could also play a role. Theoretical data suggest that B, Be, S, Pd, Cd, Os, Ir and Pt could also be involved in carbon based charge transfer reactions, but no experimental evidence substantiating this view has been found.     

A study of peptide—Peptide interactions using MALDI ion mobility o-TOF and ESI mass spectrometry

Matrix-assisted laser desorption ionization ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI-IM-oTOF MS) is evaluated as a tool for studying non-covalent complex (NCX) formation between peptides. The NCX formed between dynorphin 1-7 and Mini Gastrin I is used as a model system for comparison to previous MALDI experiments (Woods, A. S.; Huestis, M. A. J. Am. Soc. Mass Spectrom. 2001, 12, 88-96). The dynorphin 1-7/Mini Gastrin I complex is stable after more than a ms drift time through the He filled mobility cell. Furthermore, the effects of solution pH on NCX ion signal intensity is measured both by MALDI-IM-MS analysis and by nanoelectrospray mass spectrometry. When compared to the previous MALDI study this work shows that all three techniques give similar results. In addition, fragmentation can be observed from of the non-covalent complex parent ion that occurs prior to TOF mass analysis but after mobility separation, thus providing NCX composition information.     

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-muM range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower high-mass sensitivity of MCP detectors.     

A quadrupole/time-of-flight mass spectrometry study of trp-cage’s conformation

Trp-cage is a synthetic 20-residue miniprotein that uses tertiary contacts to stabilize its native conformation. NMR, circular dichroism (CD), and UV-resonance Raman spectroscopy were used to probe its energy landscape. In this quadrupole/time-of-flight study, electrospray ionization charge state distribution (CSD) and solution-phase H/D exchange are used to probe Trp-cage's tertiary structure. The CSDs of Trp-cage and its mutant provide spectra showing a pH-dependent conformation change. Solution-phase H/D exchange in 30% deuterated trifluoroethanol solution of the wild type shows increased protection of one labile hydrogen in the native state. Together, CSDs and solution-phase H/D exchange are demonstrated to constitute a simple but effective means to follow conformation changes in a small tertiary protein.     

Comparison of negative chemical ionization and electron impact ionization in gas chromatography–mass spectrometry of endocrine disrupting pesticides

The study of pesticide residues belonging to endocrine disrupting chemicals (EDCs) (23 analytes of different chemical classes – organochlorines, organophosphates, pyrethroids, dicarboximides, phtalamides, dinitroanilines, pyrazole, triazinone) in apple matrix with conventional capillary GC–NCI-MS (with methane as reagent gas) in comparison to EI ionization is presented. For sample preparation QuEChERS method was applied. The lowest calibration levels (LCLs) for all pesticides were determined in both modes. Calibration in the NCI mode was performed at the concentration levels from 0.1 to 500 μg kg⁻¹ (R² > 0.999) and for EI in the range from 5 to 500 μg kg⁻¹ (R² > 0.99). From LCLs the instrumental limits of detection (LODs) and quantification (LOQs) were calculated. Chemometric study of pesticide signals in two MS modes was performed. Repeatability of all measurements, expressed by the relative standard deviations of absolute peak areas was better than 10% for the majority of compounds. Significantly lower values were obtained for the NCI mode.     

Discrimination among geometrical isomers of α-linolenic acid methyl ester using low energy electron ionization mass spectrometry

There is a consensus that electron impact ionization mass spectrometry is not capable of discriminating among geometrical isomers of unsaturated fatty acid methyl esters (and in general olefinic compounds). In this paper, we report the identification of all eight geometrical isomers of α-linolenic acid, one of the few essential ω-3 fatty acids that has attracted great attention, using low-energy electron ionization mass spectrometry. Three electron energies 70, 50, and 30 eV were studied and the mass spectrum of each isomer was obtained from the analysis of different concentrations of a standard mixture of α-linolenic acid methyl ester geometrical isomers to ensure the robustness of the method. Principal component analysis was employed to model the complex variation of m/z intensities across the isomers. Only using the data of 30 eV energy was complete differentiation among geometrical isomers observed. The unique cleavage pattern of the α-linolenic acid methyl ester isomers leading to a benzenium ion structure is discussed and general fragmentation rules are derived using the mass spectra of over 300 compounds with different kinds and levels of unsaturation. Application of the proposed method is not limited to α-linolenic acid. It can potentially be used to identify the geometrical isomers of any compounds with an olefinic chain.     

Spectrophotometric study of melting of double stranded Poly[A]·Poly[U] in the aqueous solution

Experimentally observed changes in the electronic absorption spectrum of sodium salt of synthetic polyadenyl–polyuridyl acid (Poly[A]·Poly[U]) in the aqueous solution (pH = 7.0, I = 0.15 mol/L) with the change in temperature were explained in terms of co-existence of two different forms of the Poly[A]·Poly[U] strands. Chemometrics analysis of full set of melting curves measured at 120 wavelengths in the UV spectral range has been performed using evolving factor analysis (EFA) and KALS designed for the calculation of equilibrium constants from spectrometry data without a priori set model of the system. Using the Van’t Hoff equation, we have determined thermodynamic constants of the biopolymer transition from the double stranded helix into the random coil conformation (helix–coil transition), they can be further used to quantify the possibility of these structures to be involved in biochemical processes. Molecular model of partially denatured Poly[A]′·Poly[U]′ complex has been biult by means of molecular mechanics.     

Distribution profiles of nitroxide spin probes in human skin—a combined study using spatially resolved electron spin resonance spectroscopy and mass spectrometry

Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective “ESR-silent” hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified.     

Characterization of permethylated β-cyclodextrin-peptide noncovalently bound complexes using electron capture dissociation mass spectrometry (ECD MS)

Electron capture dissociation mass spectrometry (ECD MS) was carried out for a number of β-permethylated cyclodextrin (CD)-peptide noncovalent complexes in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Examined peptides included Angiotensin II (DRVYIHPF), Substance P (RPKPQQFFGLM), and Bradykinin (RPPGFSPFR) and its analogs (PPGFSPFR and RPPGFSPF). ECD MS for doubly protonated complexes [M:CD+2H]²⁺ mainly yielded cleavage of the backbones of the constituent peptide with little disassembly of a peptide and β-CD. Analysis of ECD MS fragments indicated that a protonated basic amino-acid residue or N-terminal amino group interacted more favorably with β-CD than did aromatic group-containing amino-acid residues (inclusion complex). In contrast to the formation of inclusion CD complexes in solution, we observed no specific evidence from our ECD MS mass spectra to support the generation of phenyl inclusion complexes in the gas phase. For gas-phase peptides, we suggest that ion–dipole interaction is the main driving force for the formation of noncovalent β-CD complexes rather than phenyl inclusion interactions.     

Application of Langmuir–Blodgett technology for the analysis of saturated fatty acids using the MALDI-TOF mass spectrometry

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Investigation of the interaction of Mercurochrome® constituents with proteins using liquid chromatography/mass spectrometry

The interaction of Mercurochrome®, a medical preparation based on the mercuryorganic compound merbromin, with free thiols in low molecular weight peptides and in proteins has been investigated by means of liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). β-Lactoglobulin A (β-LGA) from bovine milk (18.4 kDa) has been used as the model protein. It was found that, in contrast to assumptions in literature, the commercial product itself is a heterogeneous mixture of moderate chemical stability, which may contain precipitated Hg salts depending on storage time and conditions. Further variability results from different degrees of bromination of the fluorescein backbone of the compound. The formation of mercury compound–protein adducts was detected. The peptide sequence T13 containing a free thiol residue was identified as the binding site for mercury species after tryptic digestion of β-lactoglobulin A. While fresh Mercurochrome® tends to the formation of a Hg(II)-β-LGA adducts due to excess Hg²⁺ in solution, investigations after precipitation of Hg salts yield Hg(merbromin)(β-LGA) as the major product.     

Study of the microbiodegradation of terpenoid resin-based varnishes from easel painting using pyrolysis–gas chromatography–mass spectrometry and gas chromatography–mass spectrometry

The alterations produced by microbiological attack on terpenoid resin-based varnishes from panel and canvas paintings have been evaluated using pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) and gas chromatography–mass spectrometry (GC–MS). The proposed methods include the on-line derivatisation of drying oils and diterpenoid resins using hexamethyldisilazane during pyrolysis and the application of methyl chloroformate as a derivatisation reagent for triterpenoid resins in GC–MS. Two types of specimens, consisting of model oil medium prepared from linseed oil and model spirit varnishes prepared from colophony and mastic resins dissolved in turpentine, have been used as reference materials. For a series of specimens upon which different genera of bacteria and fungi were inoculated and encouraged to grow, analyses indicated that no mechanisms that commonly occur during the attack of enzymes on drying oils and terpenoid biodegraders were observed to occur in the oil medium and varnishes studied. Thus, the degradation pathways observed in the performed trials usually occur as consequence of natural ageing. Specific trials consisting of the application of biocides to uninoculated colophony varnish resulted in the identification of processes that produce undesirable degradation of the varnish due to interactions between the biocide and the varnish components. Finally, the studied biocides—Biotin, New-Des and Nipagine—generally exhibited good inhibiting effects on the microorganisms studied, although some interesting differences were found between them regarding the application method and type of biocide.     

A study on the mechanism of reaction between BNAH and chloranil:Evidence for electron transfer

The reaction between chloranil and N-benzyldihydronicotinamide(BNAH)in boratebuffer/DMF was investigated.The reaction mixture gave a strong esr signal,which is consistentwith that of chloranil anion radical,and tetrachlorohydrophenol(QH_2)and N-benzylnicotinamide(BNA~+)were obtained as the sole products.When the reaction was run in benzene solution,a greencoloured charge-transfer complex between the reactants could be isolated,which decomposed in polarsolvents to give BNA-+ and QH_2.Based on kinetic studies by esr spectroscopy by the stopped-flowtechnique,a two-step electron-transfer mechanism for the reactionis proposed in contrast to thehydride-transfer mechanism reported in the literature.     

Novel derivatisation technique for the determination of chlorophenoxy acid type herbicides by gas chromatography–mass spectrometry

The analytical detection of chlorophenoxycarboxylic-acid-type herbicides (2,4-D, dichloprop, MCPA, etc.) in environmental samples is often a problem in instrumental analysis, as these compounds containing free carboxylic groups require chemical derivatisation prior to gas chromatographic (GC) methods. Nine chlorophenoxy-acid-type herbicide active ingredients have been derivatised successfully with trimethylsilyl N,N-dimethyl carbamate and t-butyldimethylsilyl N,N-dimethyl carbamate by forming their trimethylsilyl (TMS) and t-butyldimethylsilyl (TBDMS) esters, respectively. The detection and determination of the derivatives were performed by capillary gas chromatography–mass spectrometry. The study included determination of retention indices, mass spectral properties and comparison of derivatives produced. The mass spectra of TBDMS derivatives are usually dominated by very characteristic ions [M-57]⁺ resulting from the cleavage of t-butyl moiety during electron impact (EI) ionisation in the mass spectrometer. Limits of detection were 5 to 100 pg applying GC with EI-MS detection in full scan mode. The method, using SPE sample preparation, was applied for the analysis of 115 ground water and surface water samples collected in Békés County, Hungary in 2009.     

A concept study on non-targeted screening for chemical contaminants in food using liquid chromatography–mass spectrometry in combination with a metabolomics approach

A generic method to screen for new or unexpected contaminants at ppm levels in food has been developed. The method comprises an acidic acetonitrile extraction, detection with ultra-high-pressure liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry and statistical evaluation using a metabolomics approach comparing suspected contaminated food with uncontaminated foods. The method was tested for 26 model contaminants from 100 μg/g down to 0.4 μg/g in three brands of fresh orange juice. Blinded statistical evaluation revealed signals from all added contaminants detectable by liquid chromatography–electrospray ionisation using positive ionisation mode, while only two false-positive signals were reported. The method is primarily intended to be used for investigation of food samples suspected to be contaminated with unknown substances. Additionally it could be used to continuously monitor for appearance of new food contaminants as a complement to the specific targeted analysis that is today’s foundation of food safety analysis.     

Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry

Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.     

Energy transfer or electron transfer?—DFT study on the mechanism of [2+2] cycloadditions induced by visible light photocatalysts

The intramolecular [2+2] cycloaddition of 1,3-dienes under visible light irradiation investigated by Yoon and his co-workers shows remarkably high yield and stereoselective differences under different photocatalysts. The reaction was speculated to be induced by energy transfer. However, the origin for these phenomena is still unclear. In this scene, the detailed mechanism for the [2+2] cycloaddition of 1,3-dienes under visible light has been investigated using density functional theory B3LYP and TPSSTPSS methods. The result shows that the reaction not only can be induced by energy transfer between photocatalysts and reactants, but also can be induced by electron transfer between them. The [2+2] cycloaddition induced by energy transfer is carried out along the potential energy surface (PES) of triplet excited states (T₁) firstly, and then goes back to the singlet ground state (S₀) via MECPs (minimum energy crossing points) between the PESs of the S₀ and T₁ states, forming the product in the S₀ state. The [2+2] reaction induced by electron transfer proceeds along the doublet state PES of the cation radical reactant and the neutral four-membered ring product could be obtained by electron transfer from the corresponding reactant or reduced photocatalyst. The origin of stereoselectivity of the [2+2] reaction is attributed to the reaction mechanism difference under different photocatalysts.     

Detailed study of polystyrene solubility using pyrolysis–gas chromatography–mass spectrometry and combination with size-exclusion chromatography

Measuring polymer solubility accurately and precisely is challenging. This is especially true at unfavourable solvent compositions, when only very small amounts of polymer dissolve. In this paper, pyrolysis–gas chromatography–mass spectrometry (Py-GC-MS) is demonstrated to be much more informative and sensitive than conventional methods, such as ultraviolet spectroscopy. By using a programmed-temperature-vapourisation injector as the pyrolysis chamber, we demonstrate that Py-GC-MS can cover up to five orders of magnitude in dissolved polymer concentrations. For polystyrene, a detection limit of 1 ng mL^?1 is attained. Dissolution in poor solvents is demonstrated to be discriminating in terms of the analyte molecular weight. Py-GC-MS additionally can yield information on polymer composition (e.g. in case of copolymers). In combination with size-exclusion chromatography, Py-GC-MS allows us to estimate the molecular weight distributions of minute amounts of a dissolved polymer and variations therein as a function of time.