Identification of deoxynucleoside—polyaromatic hydrocarbon adducts by capillary zone electrophoresis—Continuous Flow-fast atom bombardment mass spectrometry

Capillary zone electrophoresis coupled to continuous flow-fast atom bombardment mass spectrometry is shown to have utility for the detection and characterization of adducts formed by the covalent attachment of four polyaromatic hydrocarbon (PAH) and amino-PAH compounds to deoxyguanosine. Normal scanning provided structural information for a 1.3 ng injection of a model adduct, while 1.3 ng of each of a mixture of adducts was sufficient to determine their molecular weights by monitoring the constant neutral loss of deoxyribose. Exploitation of this loss in the multiple reaction monitoring mode resulted in the detection of low picogram amounts of target adducts in mixtures.     

Evanescent fluorobiosensor for the detection of polyaromatic hydrocarbon based on DNA intercalation

A flow-injection analysis (FIA) system coupled with an evanescent wave (EW) Biosensor employing total internal reflection of fluorescence radiation (TIRF) for the detection of polyaromatic hydrocarbon that intercalates into DNA is reported. A highly fluorescent intercalator, “ethidium bromide,” has been used as the reference compound for detection. The EW Biosensor was developed according to the procedure described earlier (1,2). Data on the analysis of Naphthalene, 3-methy cholanthrene, 7,12-dimethylbenz(a)anthracene, 1,2-benzanthracene, and some standard reference materials supplied by the National Institute of Standards and Technology are reported. The relative ability of the polyaromatic hydrocarbon to displace ethidium bromide, based on the relative binding ratio, is found to be on the order of 7,12-dimethylbenz[a]anthracene > 3-methylcholanthrene > 1,2-benzanthracene > napthalene.     

A NMR identification of isomeric Meisenheimer adducts from 4-nitrobenzofurazan and 4-nitrobenzofuroxan

It has been shown by NMR spectroscopy that both 4-nitrobenzofurazan and 4-nitrobenzofuroxan give isomeric σ-adducts, which result from methoxide addition at the non-substituted 5-and 7-carbons.     

Capillary electrophoresis-electrospray ion-trap mass spectrometry for the separation of chlorophenols

Eighteen positional isomers of chlorophenols were separated by capillary electrophoresis (CE) and detected on-line by electrospray ionization ion-trap mass spectrometry (MS). Conditions for the coupling of CE to MS, e.g., the concentration of carrier electrolyte, the sheath liquid composition and the sheath gas flow-rate were optimized. Diethylmalonic acid (5 mM) at pH 7.25 and isopropanol-250 mM dimethylamine (80:20) as sheath liquid were used. The activation parameters for ion-trap mass spectrometric analysis of chlorophenols were optimized. The mass spectra, obtained for all the analytes, revealed that the [M-H]- ion was the base peak for all chlorophenols. Moreover, conditions for CE-MS-MS detection were established and [M-H-HCl]- ions were detected.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

In vitro antitumour activity of two isomeric cyclopalladiated compounds derived from benzoylbenzylidenimines

The DNA thermal stabilizing effect and antitumour properties of two diastereoisomeric cyclopalladiated compounds, Pd₂ [4-CH₃O–C₆H₄N?C(COC₆H₅)C₆H₄]₂(μ-OAc)₂ (I and II), derived from benzoylbenzylideneimines have been studied. The atropisomers containing two acetate-bridged PdL₂ units have a folded structure in boat form. The results show that both complexes interact with the DNA double helix but that compound II stabilizes the DNA more than compound I. It was also observed that the in vitro antiproliferative activity of compound II against colon (CX-1) and lung (LX-1) human tumour cells is higher than that of compound I. It is probable that the higher reactivity of compound II relative to compound I is due to the specific orientation of the benzoyl group with respect to the CO? CN chiral bond.     

Analysis of DNA adducts using high-performance separation techniques coupled to electrospray ionization mass spectrometry.

Identification and quantitation of covalent carcinogen-DNA adducts, an important class of biomarkers, is an integral goal in toxicological research. Since these adducts are commonly present at very low levels in in vivo samples, sensitive and specific analytical methodologies are imperative for accurate detection, characterization and quantitation. High-performance separations coupled to electrospray mass spectrometry (ESI-MS) provide the sensitivity and specificity required for the analysis of DNA adducts. This review provides an overview over the research conducted in this area, focusing on the application of HPLC-ESI-MS and CE-ESI-MS techniques for structural analysis and quantitation of modified nucleosides, nucleotides and oligonucleotides.     

Capillary liquid chromatography-mass spectrometry for the rapid identification and quantification of almond flavonoids

A rapid negative ion ESI high-performance capillary liquid chromatography-mass spectrometry method was developed to identify and quantify flavonoids (e.g., flavanols, flavonols, flavanones and glycosides). Fifteen standards and two varieties of almond skin extract powder (Carmel and Nonpareil) were used to demonstrate the chromatographic separation, reproducibility and accuracy of the method that employed a 150 mm x 0.3 mm ChromXP 3C18-EP-120 column. All standards eluted in less than 10 min, providing a 9-12x reduction in analysis time compared to existing methods (90-120 min). However, isomers (e.g., catechin/epicatechin and galactosides/glucosides) were not resolved and, therefore, identified and quantified collectively. RSDs for retention time and peak area reproducibility (mass spectrometry data) were <0.5% and <5.0%, respectively. Peak area reproducibility was greatly improved (from a RSD>10%) after the implementation of a low-flow metal needle in the ESI source. Quantitation by mass spectrometry also afforded a % error less than 5% for most compounds.     

Integrated microfluidic device for the separation and electrochemical detection of catechol estrogen-derived DNA adducts

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4–10 μM with r ² ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.     

Detection and identification of carcinogen-peptide adducts by nanoelectrospray tandem mass spectrometry

Nanoelectrospray (nanoES) tandem mass spectrometry was used to examine covalently modified peptides in crude enzymatic digests of human serum albumin (HSA) that had been exposed to either benzo[a]pyrene diol epoxide (B[a]PDE, 1), chrysene diol epoxide (CDE, 2), 5-methylchrysene diol epoxide (5MeCDE, 3), or benzo[g]chrysene diol epoxide (B[g]CDE, 4). The low flow rates of nanoES (～20 nL/min) allowed several MS/MS experiments to be optimized and performed on a single sample with very little sample consumption (～30 min analysis time/µL sample). Initially, nanoES was compared with conventional LC/MS/MS analysis of carcinogen-peptide adducts. For example, nanoES analysis of an unseparated digest of B[a]PDE-treated serum albumin revealed the same peptides (RRHPY and RRHPY-FYAPE) that were previously shown, by LC/MS/MS, to be adducted with B[a]PDE. In addition, nanoES could detect unstable peptide adducts that might not otherwise have been directly observable. Finally, nanoES was shown to be an effective way to screen mixtures of modified and unmodified peptides for which no chromatographic information is available.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]⁻ ion was typically observed in the MS² spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]⁻ ion in the MS² spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]⁻ ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSⁿ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.     

Application of capillary electrophoresis- electrospray-mass spectrometry to the separation and characterization of isomeric lipopolysaccharides of Neisseria meningitidis

A capillary electrophoresis-electrospray-mass spectrometry technique for the characterization of lipopolysaccharides (LPSs) was developed, permitting the separation of trace-level O-deacylated LPS isoforms for subsequent structural characterization using tandem mass spectrometry (MS/MS). The separation buffer and electrospray interface were optimized first using O-deacylated LPS samples from large-scale preparations. It was found that with microelectrospray or sheath-solution interface, we could separate LPS in anionic forms and detect them using either negative or positive ion mode MS. For negative ion detection mode MS, 30 mM morpholine with addition of 5% v/v methanol was employed as separation buffer. When positive ion detection mode MS was required, 10 mM ammonium acetate with addition of 5% methanol was used as separation buffer. The structural assignments obtained from MS/MS and capillary zone electrophoresis-electrospray-MS (CZE-ESMS) analyses enabled the identification of isomeric glycoforms. Application of this technique to the analysis of LPS from the galE mutants of Neisseria meningitidis strain BZ157 B5+ revealed the presence of isomeric glycoforms, in which the location of a functional group phosphoethanolamine was found to be in either inner core or lipid A-OH regions. The described technique was also applied to the analysis of LPS samples from the galE mutant of N. meningitidis strains F1576 A4+ and A4-. The occurrence of isomeric LPS glycoforms differing by the location or presence of neutral sugar residues, such as hexoses, can also be characterized using MS/MS.     

DNA aptamers for selective identification and separation of flame retardant chemicals

Polybrominated diphenyl ethers (PBDEs) are group of chemicals which are representative persistent organic pollutants (POPs) and used as brominated flame retardants for many consumer products. PBDEs were phased out since 2009 but are still frequently observed in various environmental matrices and human body. Here, we report ssDNA aptamers which bind to BDE47, one of the PBDE congeners commonly found in various environmental matrices, and show affinity to other major tri-to hepta- BDE congeners. The PBDE specific aptamers were isolated from random library of ssDNA using Mag-SELEX. Two out of 15 sequences, based on their alignment and hairpin loop structures, were chosen to determine dissociation constant with BDE47 and showed from picomolar to nanomolar affinities (200 pM and 1.53 nM). The aptamers displayed high selectivity to the original target, BDE47, and implying general specificity to PBDE backbone with varying affinities to other congeners. Further, we showed that the use of two aptamers together could enhance the separation efficiency of BDE47 and other BDE congeners when dissolved in a solvent compared to use of single aptamer. These aptamers are expected to provide a tool for preliminary screening or quick separation of PBDEs in environmental samples prior to trace quantitative analysis.     

Use of a fritless dual tapered column and a low flow interface for capillary electrochromatography-mass spectrometry

To avoid problems associated with the use of sintered frits to retain packing material, tapered columns were investigated for use with capillary electrochromatography-mass spectrometry (CEC-MS) analysis. Taking the advantage that negatively charged stationary phase particles have a net velocity directed towards the buffer reservoir (inlet) over a wide range in pH, a fritless CEC column with a single taper tip was prepared for CEC-MS analysis. During CEC-MS analysis, the tapered end was immersed in the buffer reservoir and the unmodified end was pointed toward the ionization source. For better sensitivity, this single tapered CEC column was coupled to ESI/MS using a low flow sheath liquid interface. With this setup, occasional blockage of the ESI sprayer by stationary phase particles was observed. In addition, significant dead volume was observed because the unmodified tip could not be inserted into the very end of the sprayer of the low flow sheath liquid interface. To circumvent these problems, a dual tapered CEC column was prepared. This fritless dual tapered column CEC-MS approach alleviated the problems of frit, sprayer blockage and extensive dead volume.     

Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.