Quantitative DNA-methylation in Daphnia magna and effects of multigeneration Zn exposure.

Little research on the epigenetic phenomenon of DNA methylation has been performed with invertebrates. However, a few studies have shown that effects of transient chemical exposure can be transferred to non-exposed generations through epigenetic inheritance. If this is a wide-spread phenomenon it may have major consequences for current ecological risk assessments. The presence of DNA methylation in Daphnia magna has only very recently been demonstrated, although not quantified. In the present study, the overall content of 5-methyl-2'-deoxycytidine (5 mdC) in the DNA of D. magna was determined. Additionally, the effect of transgenerational Zn exposure on the 5 mdC content was assessed. An optimized LC-MS-MS method proved to be very suitable for measuring overall levels of 5 mdC in relatively small samples of D. magna DNA. The average [5 mdC]/[dG] ratio ranged from 0.13% to 0.81%. A slight but significant decrease in DNA methylation was found in the offspring (F1) of Zn exposed daphnids in the parental generation. However, this effect did not propagate into the next generation (F2). The presence of DNA methylation in D. magna and the finding that different exposure histories can entail different levels of methylation, open the way for further investigations on the implications of epigenetics in aquatic toxicology.

Online immobilized metal affinity chromatography/mass spectrometric analysis of changes elicited by cCMP in the murine brain phosphoproteome.

An automated online immobilized metal affinity chromatography/high-performance liquid chromatography mass spectrometric (IMAC-HPLC/MS/MS) method was developed to study cytidine 3',5'-cyclic monophosphate (cCMP)-specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP.

Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?

A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry.

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.

Urinary modified nucleosides as tumor markers.

Extracts of urinary nucleosides have been sequentially purified and examined by mass spectrometric analysis. Seventeen modified nucleosides have been unequivocally identified and a further five provisionally identified. While several nucleosides were found only in a small number of extracts, the occurrence and levels of others were found to correlate with the tumour type and stage.

Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry.

For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 > m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2'-deoxyguanosine (dGuo) adduct of Benzo[a]pyrene.

Short capillary ion-pair high-performance liquid chromatography coupled to electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono-, di- and triphosphates.

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.

Analysis of urinary nucleosides. II. Comparison of mass spectrometric methods for the analysis of urinary nucleosides.

Qualitative and quantitative analyses of urinary nucleosides have diagnostic potential as tumour markers. We have developed separation techniques linked to mass spectrometric detection in order to overcome the problems associated with past identification and quantitation methods. The three methods of analysis utilised were: gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/ion-trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadrupole mass spectrometry (CapLC/TQMS). Here we compare the relative effectiveness of each of the techniques for subsequent application in the systematic study of urinary nucleoside profiles in cancer patients. All three methods proved to be valuable techniques for such urinary nucleoside analyses, and a combination rather than one single choice is concluded as the ideal.

Equilenin-2'-deoxynucleoside adducts: analysis with nano-liquid chromatography coupled to nano-electrospray tandem mass spectrometry.

The interaction of 4-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupling of miniaturized liquid chromatography (LC) to electrospray tandem mass spectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reaction of 4-hydroxyequilenin with the main 2'-deoxynucleosides were separated on a Hypersyl C(18) BDS nano-HPLC column (15 cm x 75 microm i.d.) at a flow-rate of 300 nl min(-1) using gradient elution with CH(3)OH--0.2% CH(3)COOH in H(2)O. The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution low-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts were found. The nano-HPLC column-switching ES-MS system was tested for its sensitivity on a triple-quadrupole instrument, and detection limits down to 197 fg in the single reaction monitoring mode were obtained for semi-preparatively isolated equilenin--2'-deoxyguanosine adduct.

Comparison between capillary and nano liquid chromatography-electrospray mass spectrometry for the analysis of minor DNA-melphalan adducts.

Nano liquid chromatography (nanoLC) coupled to electrospray mass spectrometry (ES-MS) was evaluated for the analysis of DNA adducts in melphalan-treated Jurkat cells. The detection limit of the nanoLC-ES-MS-MS system was assessed using a dAMP-melphalan adduct. Compared to capillary liquid chromatography (capLC) ES-MS the absolute detection limit could be improved by a factor 10, leading to the detection of 395 fg dAMP-melphalan adduct under single-ion monitoring conditions at a S/N of 14. Minor adducts such as cross-linked adducts could be detected in in vitro solutions of 2'-deoxynucleotides (dNMP) treated with melphalan using column-switching nanoLC-ES-MS. These adducts were not found using capLC-ES-MS. More detailed structural information of the alkylation sites was obtained by examining the nanoLC-ES-MS-MS data. Jurkat cells were treated with melphalan, the modified DNA was isolated and enzymatically hydrolyzed. Several modified dinucleotides were identified, the most abundant adducts were pdG(Mel(Cl))pdC (m/z=453, t(r)=17.0 min) and pdG(Mel(OH)) pdC ring opened (m/z=453, t(r)=39.5 min).

Differentiation between isomeric phenylglycidyl ether adducts of 2'-deoxyguanosine and 2'-deoxyguanosine-5'-monophosphate using liquid chromatography/electrospray tandem mass spectrometry.

The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. 2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.

Analysis of melphalan adducts of 2'-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography-electrospray tandem mass spectrometry.

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

Analysis of the DNA adducts of phenyl glycidyl ether in a calf thymus DNA hydrolysate by capillary zone electrophoresis-electrospray mass spectrometry: evidence for phosphate alkylation.

Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products.

Narrow-bore HPLC in combination with fluorescence and electrospray mass spectrometric detection for the analysis of cocaine and metabolites in human hair.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine, cocaethylene, and benzoylecgonine is described. Using direct fluorometric detection, the procedure is particularly interesting for the routine analysis of human hair samples. In the sample preparation part, the hair samples are cut and washed and two internal standards with close structural resemblance to benzoylecgonine and cocaine as well as to cocaethylene are added. Subsequently, the hair samples are homogenized, hydrolyzed overnight in a 0.1 M HCl solution at 56 degrees C, and extracted on IST Confirm HCX solid-phase extraction columns. Chromatographic separation is achieved on a narrow-bore Hypersil BDS C18 column (125 x 2.1 mm, 3 microns) by gradient elution with an ammonium acetate buffer-methanol/acetonitrile mixture. For the fluorometric detection, excitation and emission wavelengths of 242 and 315 nm, respectively, are used. This analysis protocol affords a method of high sensitivity and specificity which has been fully evaluated and validated. The data presented show good accuracy and linearity with excellent reproducibility and recovery. Because unequivocal identity confirmation is mandatory in forensic applications, an extension of the analysis protocol was accomplished toward mass spectrometric detection. We succeeded in a simple methodological transfer from LC/FL to LC/ESI-MS/MS, thus providing two complementary approaches after a single, common sample-processing step. Hair samples from 29 fatalities, all known drug users and suspected victims from a drug overdose, were analyzed in this way. Of the investigated samples, 12 were positive and the concentrations found range from 0.98 to 938 ng/mg of hair for cocaine and from 1.45 to 388 ng/mg of hair for benzoylecgonine. Traces of cocaethylene were also found in two of the hair samples. The results obtained with LC/ESI-MS/MS were in close agreement with those obtained with LC/FL, positively confirming the isolates' identity and structure by means of the resulting MS/MS spectra.

Analysis of the DNA damage induced by phenyl glycidyl ether using capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation between phenyl glycidyl ether (PGE) and calf thymus DNA was investigated. Agarose slab gel electrophoresis of DNA incubated with PGE revealed that nearly all high-molecular-weight species were degraded after 10 h of incubation. After DNA precipitation the reaction products present in the supernatant were subjected to a solid-phase extraction on a polystyrene divinylbenzene copolymer, enabling analysis on capillary zone electrophoresis (CZE), using sample stacking. These reaction products were mainly produced during the first 10 h of incubation, indicating that these products result from the DNA degradation. On the other hand, analysis of the adducts present in the enzymatic digest of the DNA pellet revealed that these adducts were formed only after 10 h of incubation. The reaction products present in the DNA supernatant were identified by on-line coupling of CZE to electrospray tandem mass spectrometry. Three major reaction products resulted from phosphate alkylation, as proven by the analysis of the corresponding low-energy CAD product ion mass spectra. This phosphate alkylation results in phosphotriesters which readily hydrolyze, resulting in DNA strand breaks.

Development of a nanoscale liquid chromatography/electrospray mass spectrometry methodology for the detection and identification of DNA adducts.

In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.

Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2'-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified 2'-deoxynucleotide adducts were analyzed by capillary zone electrophoresis (CZE), CZE-electrospray mass spectrometry (CZE/ES-MS) and CZE-electrospray tandem mass spectrometry (CZE/ES-MS/MS) using sample stacking. For the CZE analyses, a homemade system was developed in order to enhance the reproducibility of the retention times. This modification enabled the total comparison of the electropherograms obtained for the analysis of 2'deoxynucleotides mixtures with the electropherograms obtained for the DNA hydrolysates both treated with PGEs. The assignment of adducted and nonadducted 2'-deoxynucleotide peaks was unambiguous. Analysis of the CZE/ES-MS data gave the necessary structural information and revealed the presence of mono- and dialkylated 2'-deoxynucleotides. Interpretation of the CZE/ES-MS/MS data of the monoalkylated products allowed differentiation between purine or pyrimidine alkylation and alkylation of the 5-phosphate moiety. Recording of full-scan mass spectra during CZE/ES-MS/MS analysis of 2'-deoxynucleotide reaction mixtures and DNA hydrolysates was possible, using the described CZE sample stacking technique.