The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide‐oligonucleotide adducts determined using nanoLC/MS/MS

Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on‐line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, ( ± )‐(7R,8S,9S,10R)‐benzo[a]pyrene 7,8‐diol 9,10‐epoxide [( ± )‐anti‐benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14‐mer double stranded oligonucleotide (5′‐ACCCG₅CG₇TCCG₁₁CG₁₃C‐3′/5′‐GCGCGGGCGCGGGT‐3′) derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational ‘hot spots’ and have also been reported as chemical ‘hot spots’ for the formation of BPDE‐DNA adducts. In evaluating the effect of cytosine methylation on BPDE‐DNA adduct binding, it was found that codon 156, containing the nucleobase G₅ instead of the mutational hot spot codons 157 (G₇) and 158 (G₁₁), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G₅? G₁₁ > G₁₃ > G₇. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G₁₁ CpG site (codon 158) approximately two‐fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G₅? G₇ > G₁₁ > G₁₃. The three‐ to four‐fold increase in adduction at the mutational hot spot codon 157 (G₇) relative to the unmethylated or single‐stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run. Copyright © 2009 John Wiley & Sons, Ltd.     

Reactivity of guanine at m5CpG steps in DNA: evidence for electronic effects transmitted through the base pairs.

BACKGROUND: Mitomycin C (MC), a DNA cross-linking and alkylating agent, targets guanines in the m5CpG sequence with 2-3-fold preference over guanines in unmethylated CpG. Benzo[a]pyrenediolepoxide (BPDE) and several other aromatic carcinogens form guanine adducts with an identical selectivity for m5CpG, and in certain cancers G to T transversion mutation 'hotspots' in the p53 tumor suppressor gene are more frequent at this sequence than at guanines in other sequences. MC appears suitable to probe the general mechanism of this selectivity. RESULTS: A 162-bp DNA fragment containing C, m5C or f5C (5-fluoro cytosine) at all cytosine positions was cross-linked by MC at guanines in CpG steps. The extent of cross-linking increased in the order f5C < C < m5C. Monoalkylation or cross-linking of duplex 12-mer oligonucleotides containing a single CpG, f5CpG or m5CpG step gave yields of adducts that increased in the same order. The rates showed a correlation with the Hammett sigma constant of the methyl and fluoro substituents of the cytosine. Only the base-pair cytosine substituent influenced reactivity of guanine. CONCLUSIONS: The 2-amino group of guanine in the m5CpG sequence of DNA has a greater nucleophilic reactivity with mitomycin than CpG. Evidence is presented for a novel mechanism: transmission of the electron-donating effect of the 5-methyl substituent of the cytosine to guanine through H-bonding of the m5C.G base pair. The results explain the enhanced reaction of BPDE at m5CpG in DNA and the origin of G-T mutational hotspots in the p53 gene in cancer.     

FLUORESCENCE STUDY OF THE PHYSICO-CHEMICAL PROPERTIES OF A BENZO(A)PYRENE7,8-DIHYDRODIOL9,10-OXIDE DERIVATIVE BOUND COVALENTLY TO DNA

Abstract— Covalent complexes between 7 ,8-dihydrodiol 9.10-oxide benzo(a)pyrene (BPDE) and DNA with a modification of one BPDE molecule per 1000 DNA bases were prepared in vitro . The same stereoselective and chemically homogeneous binding of BPDE to native DNA was observed, as reported earlier for human and bovine bronchial explants. The fluorescence of the pyrene-like aromatic moiety of BPDE bound to DNA in vitro was used as a probe of the microenvironment of the BPDE molecule in order to obtain information about the structure of the BPDE-DNA complex dissolved in aqueous solution. Fluorescence techniques, based on the quenching of the singlet excited states by metal ions such as Ag⁺, by iodide ions, and by molecular oxygen are described, which provide a method for differentiating between external and internal (intercalation) binding of polycyclic aromatic molecules to DNA. Silver ions, which bind specifically to DNA bases, exhibit a strong quenching effect on noncovalently bound, intercalated benzo(a)pyrene; on the other hand, there is no quenching effect on the fluorescence of BPDE in the covalent DNA adduct. Quenchers such as O₂ and iodide ions, which do not specifically bind to DNA and are dissolved in the solution external to the DNA molecule, exhibit a quenching effect on the BPDE chromophore. Furthermore, the fluorescence yield of the BPDE-DNA complex decreases with increasing DNA concentration, an effect which is not observed with non-covalently bound intercalated benzo(a)pyrene-DNA complexes, and which is attributed to intermolecular DNA-DNA interactions. The results of these studies indicate that the pyrene-like chromophore in the covalent BPDE-DNA complex is not intercalated between the base pairs, and that it is located in an accessible region external to the DNA helix. Possible structures are discussed.     

Selective digestion and novel cleanup techniques for detection of benzo[a]pyrene diol epoxide-DNA adducts by capillary electrophoresis/mass spectrometry

Benzo[a]pyrene (BP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) which, upon metabolic conversion to reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), has been found to attach covalently to DNA. Given the low level of DNA adducts typically present in vivo or in vitro, an essential first step prior to capillary electrophoresis/mass spectrometry (CE/MS) (or liquid chromatography/mass spectrometry (LC/MS)) analysis of the DNA digests is the removal of the bulk non-adducted nucleotides, enzymes or salts, and isolation of enriched adducts. This report focuses on the development of novel sample handling methods aimed at facilitating the analysis of BPDE-DNA adducts by CE/MS. This approach involves a simple variation on the digestion procedure, in combination with the use of metal affinity ZipTips for the more efficient cleanup of BPDE-DNA adducts formed in vitro for subsequent CE/MS analysis. The previously described digestion procedure, consisting of micrococcal nuclease, spleen phosphodiesterase and nuclease P1, allows for selective dephosphorylation of normal nucleotides, while leaving adducted nucleotides intact. Metal affinity ZipTips, typically used for selective extraction of phosphopeptides, were used here for extraction of adducted nucleotides. The utility of metal affinity SPE was tested on mixtures of dG and dGp, wherein nucleotide extracts contained no detectable nucleosides by CE/UV analysis. An in vitro BPDE-DNA incubation was then digested using the above procedure. Metal affinity solid-phase extraction (SPE) was subsequently used for the selective isolation of phosphorylated components, i.e., adducted nucleotides, from the mixture of enzymes and non-adducted nucleosides. SPE extracts were enriched in nucleotide adducts and analyzed using sample stacking and CE/MS. This method has several advantages over previously described cleanup procedures for dGp-BPDE adducts: fast, simple, uses commercially available materials, no need for excessive dilution (small scale), the suitability for use with automation, and possible applicability to other bulky hydrophobic adducts.     

Computational investigation of thermochemical properties of non-natural C-nucloebases: different hydrogen-bonding preferences for non-natural Watson–Crick base pairs

In the present density functional theory study, we have compared intrinsic properties of non-natural nucleobases (acA, acG, acC, and acT nucleobases) such as proton affinities, gas phase acidities, tautomerization, and hydrogen-bonding properties with those in normal Watson–Crick nucleobases (A, G, C, T nucleobases). The hydrogen-bonding interactions in non-natural and Watson–Crick base pairs were studied at B3LYP/6-311++G (d,p) level regarding their geometries, energies, and topological features of the electron density. The quantum theory of atoms-in-molecule (QTAIM) and natural bond orbital (NBO) analyses were employed to elucidate the interaction characteristics in base pairs. The electron density ρ(r) as well as its Laplacian $ \nabla^{2} $ ρ(r) at the hydrogen bond critical point predicted by QTAIM is strongly correlated with hydrogen bond structural parameter and the second-order perturbation energies in NBO scheme. Our results show that most of hydrogen bonds in normal Watson–Crick and non-natural base pairs must be considered as electrostatic interactions. Results of calculations revealed that energetic values of hydrogen bonds in T–A base pair are more than those in ac T–ac A base pair, while values of hydrogen bonds in C–G base pair and ac C–ac G base pair are almost the same. These results confirmed stability order of stabilization energies of these base pairs.     

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.     

CE of Small DNA Fragments Using Linear Polyacrylamide Matrices

Mutations at codons 248 and 249 of p53 gene showed a relatively high incidence in gastric cancer patients. Development of novel methods for the detection of codon mutations is of great importance for gastric cancer research. Studies have showed that the separation matrix can significantly influence the separation efficiency and resolution of small DNA fragments in CE. In order to achieve baseline separation of PCR-amplified products of small DNA fragments from gastric cancer tissue, linear polyacrylamides (LPA I and LPAII) were designed and synthesized in the current study. LPAI and LPAII were used as separation matrixes to separate small size fragments (less than 70 bp) of pBR322/BsuRI DNA Marker and the separation conditions were optimized. Optimum separations were performed at 25 kV in reversed-polarity mode with capillary temperature set at 15 °C. The signal of DNA fragments was detected using laser-induced fluorescence detector, with an argon ion laser as the excitation source that emits at 488 nm. A 520 nm bandpass filter was used as an emission cut-off filter. The resolution of small DNA fragments was higher when LPAI was used as separation matrix compared to LPAII, accompanied with longer migration time. The results indicated that LPAI as separation matrix was more efficient for the separation of small DNA fragments (less than 70 bp) than other LPAs. A rapid and sensitive analysis method for the separation and detection of small DNA fragments (less than 70 bp) was established in this study. The method was successfully applied to detect the mutations at codons 248 and 249 in p53 gene from gastric cancer tissues.     

Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)₂ was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.     

Specific Purine‐Purine Base Pairing in Linear Alanyl‐Peptide Nucleic Acids

Purine‐purine base pairing with guanine, isoguanine, 2,6‐diaminopurine, and xanthine is investigated within the topology of alanyl‐PNA. Alanyl‐PNA is based on a regular peptide backbone with alternating configuration of the amino acids. The nucleobases are covalently linked as side chains. Their distance in peptides with β‐sheet conformation is similar to the favored base‐pair stacking distance. Therefore, alanyl‐PNA provides self‐pairing linear double‐strands. The linear double‐strand topology does not restrict base‐pair size and geometry. The favored base pairs are formed mostly dependent on recognition by H‐bonding. The synthesis of the nucleo‐amino acids with unnatural nucleobases and their oligomerization is described. Hexamers and a tetramer based on 2,6‐diaminopurine‐xanthine and guanine‐isoguanine base pairs were observed with very high stabilities. For xanthine‐xanthine self‐pairing, an unusual tridentate reverse Watson‐Crick pairing mode is indicated, that is only possible with xanthines pairing in different tautomeric forms. To investigate the nature of xanthine‐xanthine base pairs in more detail, quantum‐chemical calculations were performed. They establish the easier tautomerization of xanthine compared to uracil and indicate that, in the AO basis‐set limit, the tridentate pairing mode with mixed tautomers is favored.     

p53 gene mutations in SKH-1 mouse tumors differentially induced by UVB and combined subcarcinogenic benzo[a]pyrene and UVA

We compared the frequency and spectra of p53 mutations in skin tumors from UVB-irradiated and benzo(a)pyrene-UVA-treated SKH-1 mice. Analysis of p53 mutations using a combination of polymerase chain reaction, denaturing high-performance liquid chromatography, and sequencing shows that the frequency and spectrum of p53 mutations in BaP-UVA-induced tumors are quite different from those in UVB-induced tumors. SKH-1 mice were treated with BaP-UVA or UVB for 30 weeks after which skin tumors were collected for analysis of p53 mutations. Among the 11 BaP-UVA-induced tumors with diameters of 5-10 mm, two displayed mutations in exon 8 yielding a mutation frequency of 18.2%. In contrast, the mutation frequency among BaP-UVA-induced tumors was 10.5%. In UVB-induced tumors, the mutation frequency in exon 8 was highly correlated with tumor size. A total of 77.8% of tumors with diameters larger than 10 mm contained p53 mutations. The overall mutation frequency among UVB-induced tumors was 17.9% in exon 8 and only 3.8% in exon 5. Hotspots for p53 mutation in UVB-induced tumors were found at codons 128 and 149 (exon 5), and at codons 268, 270, 271 and 273-276 (exon 8). In addition to widely recognized C-->T missense mutations, there were also tandem CC-->AG changes coupled with either an insertion of T, a C-->G substitution or G-->C/T mutations. All of the mutations were found at tri- or tetra-pyrimidine sites. Thirty-nine per cent of all p53 mutations occurred at codons 274 and 275; 53% occurred at codons 268-271. Two multiple mutation clusters were located at codons 268-271 and 274-276. Both BaP-UVA and UVB caused C-->T transitions at codon 275 in exon 8. A C-->T mutation at codon 294 was induced only by BaP-UVA treatment. In contrast to UVB treatment, BaP-UVA treatment did not induce any mutations in exon 5. We show that individually subcarcinogenic levels of BaP and UVA synergistically induce a novel p53-mutation fingerprint. This fingerprint could serve as a prognostic indicator for the development of BaP-UVA-induced skin tumors.     

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.     

Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column‐switching liquid chromatography/tandem mass spectrometry

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software‐based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH‐modified DNA samples were obtained by reaction of the anti‐dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2′‐deoxynucleosides. Positive LC/electrospray ionisation (ESI)‐MS/MS collision‐induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2′‐deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2′‐deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH‐adducted 2′‐deoxynucleosides was achieved using online column‐switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H]⁺ to [M+H–116]⁺ transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H–116]⁺ transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods. Copyright © 2010 John Wiley & Sons, Ltd.     

Stereocontrolled protein surface recognition using chiral oligoamide proteomimetic foldamers

The development of foldamers capable of selective molecular recognition of solvent exposed protein surfaces represents an outstanding challenge in supramolecular chemical biology. Here we introduce an oligoamide foldamer with well-defined conformation that bears all the hallmarks of an information rich oligomer. Specifically, the foldamer recognizes its target protein hDM2 leading to inhibition of its protein–protein interaction with p53 in a manner that depends upon the composition, spatial projection and stereochemistry of functional groups appended to the scaffold. Most significantly, selective inhibition of p53/hDM2 can be achieved against four other targets and the selectivity for p53/hDM2 inhibition versus Mcl-1/NOXA-B inhibition is critically dependent upon the stereochemistry of the helix mimetic.     

A Simple LC-MS/MS Method for the Quantification of PDA-66 in Human Plasma

The treatment of cancer is one of the most important pharmacotherapeutic challenges. To this end, chemotherapy has for some time been complemented by targeted therapies against specific structures. PDA-66, a structural analogue of the inhibitor of serine–threonine kinase glycogen synthase kinase 3β SB216763, has shown preclinical antitumour effects in various cell lines, with the key pathways of its anticancer activity being cell cycle modulation, DNA replication and p53 signalling. For the monitoring of anticancer drug treatment in the context of therapeutic drug monitoring, the determination of plasma concentrations is essential, for which an LC-MS/MS method is particularly suitable. In the present study, a sensitive LC-MS/MS method for the quantification of the potential anticancer drug PDA-66 in human plasma with a lower limit of quantification of 2.5 nM is presented. The method was successfully validated and tested for the determination of PDA-66 in mouse plasma and sera.     

Quantitative determination of major platelet activating factors from human plasma

Platelet activating factor (PAF) is a potent lipid mediator that is involved in many important biological functions, including platelet aggregation and neuronal differentiation. Although an ELISA assay has been used to measure PAF levels, it cannot distinguish between its isoforms. To achieve this, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used instead. However, isobaric lysophosphatidylcholine (lyso PC), which is often present in large amounts in complex biological samples and has similar retention times in many LC conditions, can affect the accurate measurement of PAF. The present study examined the fragmentation behavior of major PAF and lyso PC during various MS/MS conditions. Fragment ions at m/z 184 and at m/z 104 were abundantly observed from MS/MS of lyso PCs. PAF provided a dominant fragment ion at m/z 184, but a fragment ion at m/z 104 was almost never produced, regardless of the collision energy. Thus, the two fragment ions at m/z 184 and m/z 104 were used to accurately measure PAF levels. First, the fragment ion at m/z 184 and the retention time of PAF in LC-MS/MS were used to identify and quantitate PAF. However, if there were small retention time shifts, which are common in multiple sample runs, and lipid composition in a sample is very complicated, the fragment ion at m/z 104 was used to confirm whether the fragment ion at m/z 184 belonged to PAF. This novel method accurately determined the major PAF (C16:0 PAF, C18:0 PAF, and C18:1 PAF) levels in human plasma.     

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T? Hg^II? T base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases C? Ag^I? C formed in an RNA duplex. One Ag^I occupies the center between two cytosines and forms a C? Ag^I? C base pair through N3? Ag^I? N3 linear coordination. The C? Ag^I? C base pair formation does not disturb the standard A‐form conformation of RNA. Since the C? Ag^I? C base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.     

Elucidating organ-specific metabolic toxicity chemistry from electrochemiluminescent enzyme/DNA arrays and bioreactor bead-LC-MS/MS

Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termed genotoxic. Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)₂(PVP)₁₀]²⁺ {(PVP = poly(4-vinylpyridine))} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.