NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI⁺-HRMS³ analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5–1.5 ppm). EB-GII concentrations increased linearly from 1.15?±?0.23 to 10.11?±?0.45 adducts per 10⁶ nucleotides in HT1080 cells treated with 0.5–10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17?±?0.05, 0.33?±?0.08, and 0.50?±?0.04 adducts per 10⁶ nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20?±?0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI⁺-HRMS³ Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

Colorimetric determination of 8-hydroxy–2′-deoxyguanosine using label-free aptamer and unmodified gold nanoparticles

We report on a sensitive and specific method for the visual and colorimetric detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in human urine by using the 8-OHdG-aptamer (Apt) as the recognition element. The aptamer was adsorbed on the surface of gold nanoparticles which enhances their stability. On addition of 8-OHdG, the conformation of the aptamer changes to form a G-quadruplex structure, thereby losing the ability to protect the nanoparticles and causing a color change from red to blue. The conformational changes were also studied by circular dichroism. The response is linearly dependent on the concentration of 8-OHdG in the range from 5.6 to 282 nM, the limit of detection is 1.7 nM, the relative standard deviation ranges from 1.1 to 4.2 % (for n?=?6), and the recoveries range from 95.9 to 104.9 %. The method paves the way for visual analysis of samples without the use of costly instruments.

Electron Capture Dissociation of Divalent Metal-adducted Sulfated N-Glycans Released from Bovine Thyroid Stimulating Hormone

Sulfated N-glycans released from bovine thyroid stimulating hormone (bTSH) were ionized with the divalent metal cations Ca²⁺, Mg²⁺, and Co by electrospray ionization (ESI). These metal-adducted species were subjected to infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) and the corresponding fragmentation patterns were compared. IRMPD generated extensive glycosidic and cross-ring cleavages, but most product ions suffered from sulfonate loss. Internal fragments were also observed, which complicated the spectra. ECD provided complementary structural information compared with IRMPD, and all observed product ions retained the sulfonate group, allowing sulfonate localization. To our knowledge, this work represents the first application of ECD towards metal-adducted sulfated N-glycans released from a glycoprotein. Due to the ability of IRMPD and ECD to provide complementary structural information, the combination of the two strategies is a promising and valuable tool for glycan structural characterization. The influence of different metal ions was also examined. Calcium adducts appeared to be the most promising species because of high sensitivity and ability to provide extensive structural information.

Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

The fragmentations of [AA + M]⁺ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag^?).

A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15 nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody–DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5 pg L^?1 to 10 ng L^?1, and the limit of detection (LOD) was 1.72 pg L^?1. Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82 % to 112 %, and the coefficient of variation (CV) was acceptable. The results were compared with GC–ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL^?1 milk and 0.4 ng mL^?1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n?=?8) or non-transgenic (n?=?7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Colorimetric detection of peroxynitrite-induced DNA damage using gold nanoparticles, and on the scavenging effects of antioxidants

We describe a rapid and convenient colorimetric method for the detection of oxidative DNA damage caused by peroxynitrite (ONOO^?) using unmodified gold nanoparticles (AuNPs). AuNPs are stable in the presence of single-stranded DNA (ssDNA) against the aggregation induced by a high ionic strength. If adsorbed ssDNA are cleaved by ONOO^? to form smaller fragments, the AuNPs rapidly aggregate due to electrostatic attraction. As a result, the color of the solution changes from red to blue, and this can be seen with bare eyes. We also have evaluated the activity of the antioxidants gallic acid, ascorbic acid and caffeic acid to scavenge ONOO^?. This method therefore also can be applied to screen for anti-oxidation drugs and agents.

Rapid and sensitive detection of bacteria via platinum-labeled antibodies and on-particle ionization and enrichment prior to MALDI-TOF mass spectrometry

We introduce a rapid and sensitive approach to study the interactions of an affinity probe with the bacterial wall. Immunoglobulin was immobilized on platinum nanoparticles, and the resulting probe nanoparticles bind to bacterial walls as confirmed by transmission electron microscopy. A MALDI-MS assay was developed that can detect ~10⁵ cfu mL^?1 of S. marcescens and E. coli. This approach enables simple, rapid and straightforward detection of bacterial proteins, with high resolution and sensitivity, and without the requirement for tedious washing/separation steps.

Impedimetric aptasensor for Staphylococcus aureus based on nanocomposite prepared from reduced graphene oxide and gold nanoparticles

We describe here an aptasensor for the ultrasensitive detection of Staphylococcus aureus by electrochemical impedance spectroscopy (EIS). Single-stranded DNA was linked to a nanocomposite prepared from reduced graphene oxide (rGO) and gold nanoparticles (AuNP). Thiolated ssDNA was covalently linked to the AuNPs linked to rGO, and probe DNA was immobilized on the surface of an AuNP-modified glassy carbon electrode to capture and concentrate Staph. aureus. The probe DNA of the aptasensor selectively captures the target bacteria in its three-dimensional space, and these results in a dramatic increase in impedance. Scanning electron microscopy, cyclic voltammetry and EIS were used to monitor the single steps of the electrode assembly process. The effect was utilized to quantify the bacteria in the concentration range from 10 to 10⁶ cfu mL^?1 and with a detection limit of 10 cfu mL^?1 (S/N?=?3). The relative standard deviation of Staphylococcus aureus detection was equal to 4.3 % (10⁵ cfu mL^?1, n?=?7). In addition to its sensitivity, the biosensor exhibits high selectivity over other pathogens.

The detection of T-Nos,a genetic element present in GMOs,by cross-priming isothermal amplification with real-time fluorescence

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10³ copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2′-deoxycytidine (5-medC) and 2′-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O ⁶-methylguanine (O ⁶-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16–4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O ⁶-meG and N3-meA were however found to be undetectable in all lung tissue samples.

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c ^m+ and z ^m+? products of cleavage at each of its molecular ion’s inter-residue bonds. For example, the ECD spectra of ubiquitin (M?+?nH)ⁿ⁺ ions, n?=?7–13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ^(1–7)+ and eight z ^(1–8)+? spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the c ^m+ or z ^m+? products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD⁺), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD⁺, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD⁺, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD⁺ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD⁺, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

Sensitive fluorescent detection of Staphylococcus aureus using nanogold linked CdTe nanocrystals as signal amplification labels

A sensitive fluorescent assay was developed for the detection of DNA specifically for Staphylococcus aureus. A sandwich-type detection system was fabricated by first immobilizing biotinylated capture DNA on avidin-modified wells of microplates, then hybridizing the capture DNA with one end of the target DNA, and then recognizing the other end of the target DNA with a signal probe labeled with CdTe nanocrystals and gold nanoparticles (Au-NPs) at the 3′- and 5′-terminus, respectively. Hybridization was monitored by measuring the fluorescent intensity of the assembly. The experimental results demonstrated that the incorporation of Au-NPs in this detection system can significantly enhance the sensitivity and the selectivity because a single Au-NP can be loaded with hundreds of signal DNA probe strands modified with CdTe nanocrystals. Under the optimized conditions, a detection limit of 10 fmol of DNA per L can be achieved and at least 50 colony forming units of Staph. aureus per mL of sample can be detected. The method was assessed by analyzing real samples, and it was validated by comparing it to an official standard method.

Detection of mismatched caspase-3 DNA oligonucleotides with an SPR biosensor following amplification by Taq polymerase

We demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus (Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50 pM, 0.1 nM, 0.7 nM, 7 nM, and 20 nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2 μM of Mg(II) and 0.3 mM of dNTP.

Electrochemical detection of in situ DNA damage induced by enzyme-catalyzed Fenton reaction. Part II in hydrophobic room temperature ionic liquid

A hydrophobic room temperature ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]), was applied as nonaqueous solvent for the generation of hydroxy radical (?OH) through glucose oxidase-catalyzed Fenton reaction. The enzyme catalyzes the oxidation of glucose, and the produced H₂O₂ further reacts with transition metal ions, generating hydroxyl radicals. They attacked DNA and led its damage. This was detected by square wave voltammetry (SWV) of the electroactive indicator Co(bpy) ₃ ³⁺ . It bound more strongly to intact DNA, and the SWV peak currents decreased at the potential of 0.064?V when DNA was damaged. The experimental results testified that the antioxidants, ascorbic acid, aloe-emodin and rutin, inhibited oxidative DNA damage by hydroxyl radicals. The method is promising for rapid, sensitive, and inexpensive detection of DNA damage.

Bioactivity fingerprint analysis of cyclooxygenase-2 ligands from radix Aconiti by ultrafiltration–UPLC–MSn

A novel fingerprinting method, bioactivity fingerprint analysis, based on an ultrafiltration–ultraperformance liquid chromatography–multistage tandem mass spectrometry (UPLC–MS ⁿ ) method is proposed for the quality control of herbal medicines from the bioactivity viewpoint concerning the efficacy of herbal medicines. The bioactivity fingerprints reflecting the anti-inflammatory activities of radix Aconiti and radix Aconiti preparata were established. With use of ultrafiltration UPLC–MS ⁿ , 11 cyclooxygenase-2 ligands from radix Aconiti preparata and 14 cyclooxygenase-2 ligands from radix Aconiti were found after incubation with cyclooxygenase-2. Twelve of the cyclooxygenase-2 ligands were identified by the ultraperformance UPLC–MS ⁿ method. The enrichment factor of each peak in the bioactivity fingerprint was calculated and was demonstrated to be characteristic, which makes bioactivity fingerprint analysis for the quality control of herbal medicines possible from the viewpoint of their bioactivities.

Electrochemical detection of in situ DNA damage induced by enzyme-catalyzed Fenton reaction. Part I: in phosphate buffer solution

We report on a biosensor for the electrochemical detection of the damage of DNA and of antioxidant protecting DNA. The biosensor was constructed by co-immobilization of DNA and glucose oxidase (GOx) on a glassy carbon electrode. Under aerobic conditions, GOx catalyzes the oxidation of glucose, and the hydrogen peroxide produced reacts with ferrous ions in a Fenton-type reaction to generate hydroxy radical. This was validated by UV–vis spectroscopy. The hydroxy radical can cause serious oxidative damage to DNA, and this can be detected by square wave voltammetry of the electroactive indicator Co(bpy) ₃ ³⁺ . The effects of pH value, incubation time, and the concentration of glucose and ferrous ion were optimized. The effects of the antioxidants ascorbic acid and aloe emodin on DNA damage were also investigated within the concentration range from 0.05 to 200?μM. This work provides an in-vitro model system to mimic the processes in oxidative DNA damage by a simple electrochemical approach.

MS/MS Fragmentation Behavior Study of meso-Phenylporphyrinoids Containing Nonpyrrolic Heterocycles and meso-Thienyl-Substituted Porphyrins

Free base and cobalt(II) complexes of six meso-tetraphenylporphyrinoids containing nonpyrrolic heterocycles and of three meso-thienylporphyrins were investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their fragmentation was studied in a quadrupole ion trap as a function of the porphyrinoid macrocycle structure and compared with the fragmentation behavior of the benchmark compound meso-tetraphenylporphyrin. In situ oxidation of the neutral cobalt(II) complexes under ESI conditions produced singly charged cobalt(III) porphyrinoid ions; the free bases were ionized by protonation. For the porphyrinoids with an intact porphyrin core, the major fragmentation pathways observed were the losses of the meso-substituent (for meso-phenyl groups) and characteristic fragmentations of one or more meso-substituents (for the meso-thienyl group). Complex fragmentation pathways were observed for porphyrinoids with modifications to the porphyrin core but chemically reasonable structures could be assigned to most fragments, thus delineating general patterns for the behavior of pyrrole-modified porphyrins under CID conditions.

Differential pulse anodic stripping voltammetric determination of Cd and Pb at a bismuth glassy carbon electrode modified with Nafion, poly(2,5-dimercapto-1,3,4-thiadiazole) and multiwalled carbon nanotubes

A simple, cheap, and nonpolluting method was developed for the cloud point extraction of gold (Au) and palladium (Pd). It is based on the complexation reaction of Au and Pd with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) and micelle mediated extraction of the complex using the non-ionic surfactant poly(ethylene glycol) mono-p-nonylphenyl ether (PONPE 7.5). Under the optimized experimental conditions, the enrichment factors are 16 and 17 for Au and Pd, respectively, for 15?mL of preconcentrated solution. The limits of detection are 3.8???g?L^?1 and 1.8???g?L^?1 for Au and Pd, respectively. The relative standard deviations are 1.4% for Au and 0.6% for Pd (n?=?11). The method was successfully applied to the determination of Au and Pd in certified reference materials and mine samples.