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.

Cobalamin as an analytical tool for analysis of oxirane metabolites of 1,3-butadiene: development and validation of the method.

The reduced form of vitamin B12 [cob(I)alamin] is known to be a supernucleophile, with the ability to react 10(5) times faster than standard nucleophiles. Procedures have been developed where cob(I)alamin is used as an analytical tool for the trapping of electrophilically reactive compounds. In the present work, a sensitive and accurate method for determination of reactive metabolites produced in vitro has been developed and validated. Diepoxybutane (DEB), a metabolite of 1,3-butadiene, was used as a model compound. The intermediate precursor 1,2-epoxybutene (EB) was incubated in a mouse liver S9 metabolic system and the formation of DEB was studied. Samples were taken at different times from the incubation mixture and added to the cob(I)alamin. The alkyl-cobalamins (alkyl-Cbl) formed were directly analysed by a miniaturized LC-MS/MS method and column switching. The assay was linear over the concentration range of 1.5-500 microM with acceptable precision and accuracy.

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.

Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio).

In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.

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).

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.

Identification and quantitation of despropionyl-bezitramide in postmortem samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the determination and quantitation of (despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method is the result from a simple methodological transfer of a liquid chromatographic method with fluorescence detection (LC-FL) previously developed in our laboratory. A liquid-liquid back-extraction procedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solvent is performed for a basic sample cleanup. N-Methyldespropionyl bezitramide is used as the internal standard. Chromatographic separation of the analytes of interest is achieved on a Hypersil ODS 5-micron column, using a 80:20 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50:50, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring mass spectrometric technique is applied. In addition, low-energy collisional-activated dissociation (CAD) product ion spectra are recorded for a few samples. Calibration graphs are prepared for blood and urine, and good linearity is achieved over a concentration range of 1-150 ng/mL. The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples at 10 and 50 ng/mL concentration levels do not exceed 10.2% and percent of targets are within 12.1%. Postmortem samples (blood, urine, stomach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequivocal conclusions about the prior intake of bezitramide by the three subjects.

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.

Study of the enzymatic degradation of vasostatin I and II and their precursor chromogranin A by dipeptidyl peptidase IV using high-performance liquid chromatography/electrospray mass spectrometry.

The interaction of dipeptidyl peptidase IV with structurally related proteins differing in chain length, namely vasostatin I and II and their precursor protein chromogranin A, was examined using high-performance liquid chromatography in combination with electrospray mass spectrometry. Suitable analytical procedures were developed involving the use of reversed-phase high-performance liquid chromatography for purification of the enzymatic degradation products and a peptide mapping procedure for evaluating the enzymatic degradation of the large precursor protein chromogranin A. While vasostatin I was found to be a substrate for dipeptidyl peptidase IV, no N-terminal cleavage of Leu-Pro could be noted for chromogranin A. With respect to vasostatin II, N-terminal degradation was only observed after degradation in the C-terminal domain to proteins containing < or = 78 amino acids. The specificity of the N-terminal release of Leu-Pro was proved by addition of a DPP IV specific inhibitor.

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma.

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

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.

Preparative capillary zone electrophoresis in combination with off-line graphite furnace atomic absorption for the analysis of DNA complexes formed by a new aminocoumarine platinum (II) compound.

Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum (II) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.

Structural, functional, and genetic characterization of Gastrophilus hemoglobin.

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.