Biomedical subjects

J C Tabet

Publications and source records attributed to J C Tabet.

At least 19 recordsLinked to original sources

PubMed2007

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry.

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.

Adenine↗

PubMed2006

MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.

Animals↗

PubMed2006

Quantification of small therapeutic proteins in plasma by liquid chromatography-tandem mass spectrometry: application to an elastase inhibitor EPI-hNE4.

LC/ESI-MS/MS is a promising alternative to immunoassays in improving the analysis of recombinant therapeutic proteins in biological fluids for toxicity and pharmacokinetics purposes. To assess the sensitivity and validation issues associated with this technique, we use here as a model EPI-hNE4, a 56-amino acid recombinant protein, and demonstrate that a method based on tandem mass spectrometry combined with liquid chromatography and electrospray interface can reach sensitivity similar to that of ELISA but without its potential cross-reactivity. For this purpose, a triple quadrupole mass spectrometer operating in positive ion and single reaction monitoring mode with transition, m/z 1040 --> 1224.5, was used for selective peak detection. Particular issues related to the internal standard, i.e., elution and ionization patterns similar to the protein without stable isotope labeling, and to analytical interference due to endogenous binding antibodies were addressed. A limit of quantification in human or monkey plasma of 5 ng/mL was reached with a sample volume of 100 microL, corresponding to 40 fmol injected into the HPLC column. Intra- and interassay precision and accuracy were below 15%. No matrix effect was detected.

Animals↗

PubMed2006

Solid ionic matrixes for direct tissue analysis and MALDI imaging.

Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures (alpha-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and alpha-cyano-4-hydroxycinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in term of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.

Animals↗

PubMed2005

Delayed extraction experiments using a repulsing potential before ion extraction: evidence of non-covalent clusters as ion precursor in UV matrix-assisted laser desorption/ionization. Part II--Dynamic effects with alpha-cyano-4-hydroxycinnamic acid matrix.

Delayed extraction experiments were undertaken to gain a better insight into the dynamic effects involved in the ion formation in UV matrix-assisted laser desorption/ionization. Part I1 was devoted to a 2,5-dihydroxybenzoic (2,5-DHB) matrix. The results clearly demonstrated the existence and the role of high-mass precursors corresponding to a non-covalent matrix-analyte association in ion formation. In this complementary study, ion flight time and abundance were studied as a function of the delay extraction time using the matrix alpha-cyano-4-hydroxycinnamic acid (HCCA). Under our instrumental conditions, where ejected ions experienced a low repulsing electric field before extraction, two main results were obtained: (i) two ion components are observed in the peak profiles depending on the repulsing field, a first, major component (I) similar to that observed for 2,5-DHB and a second, minor component (II) apparently triggered by the delayed extraction pulse, and (ii) ion time-of-flight variation vs delay time remained lower than that noted with 2,5-DHB matrix, indicating that the initial axial velocity is smaller. The initial kinetic energy of matrix and low molecular mass peptide ions for the component I is not high enough to overcome the repulsing potential in the delay time range (200-2200 ns) and we have to assume that ions have non-covalent clusters as precursors. Complete desolvation of these clusters-aggregates would be achieved through the extraction step. Simulations of the ion time-of-flight as a function of the delay time allow the determination of the average size of the precursors, typically 4500, 40000 and 50000 u for HCCA, ACTH 7-38 and bovine insulin quasi-molecular ion, respectively, assuming that the precursors are singly charged. The size of these ion precursors is greater than that of those generated for 2,5-DHB. For component II, ions are probably not solvated and they are directly desorbed from the target. Taking into account the results on HCCA and 2,5-DHB matrices and other results from the literature, a general model for ion formation based on clusters as ion precursors is proposed and discussed.

Coumaric Acids↗

PubMed2003

Fluorinated matrix approach for the characterization of hydrophobic perfluoropolyethers by matrix-assisted laser desorption/ionization time-of-flight MS.

Characterization of fluorinated polymers in MALDI is often unsuccessful because commonly used matrixes, such as 2,5-dihydroxybenzoic acid, Indole acrylic acid, alpha-cyano-4-hydroxycinnamic acid, etc., do not desorb/ionize fluorinated polymers efficiently. This could be in part attributed to the unfavorable interaction between the matrix molecules and fluorinated oligomers due to differences in their hydrophobicities. Moreover, the relative cation affinity between the matrix molecules and the fluorinated oligomers may not favor the gas-phase cationization process of the fluorinated oligomers. To overcome these limitations, fluorinated derivates of benzoic acid (pentafluorobenzoic acid) and cinnamic acid (Pentafluoro cinnamic acid) were employed for the desorption/ionization of perfluoropolyethers. Presence of fluorine atoms in the matrix might improve the interaction between the matrix and perfluoroether during the crystallization or ionization step. With a pentafluorobenzoic acid matrix, intact silver cationized oligomers were desorbed, whereas with a pentafluorocinnamic acid matrix, loss of end group was observed. This loss could be rationalized by the dissociation of the silver cationized oligomers via an ion-dipole mechanism. This work shows the possibility of characterizing yet another important class of fluorinated polymer by MALDI-TOFMS.

Journal Article↗

PubMed2003

Preliminary study of the stability of calix[4]arene crown compounds under radiolysis.

The extraction of (135)Cs from high activity waste arising from reprocessing of spent fuel can be achieved by using calix[4]arene crown compounds. The radiolytic degradation of calix[4]arene crowns as well as their solvent, o-nitrophenyloctyl ether (NPOE), was studied using electrospray ionization mass spectrometry (ESI-MS) (that formed Cs(+) or Na(+) adducts) in nitric acid, as well as by chemical ionization tandem mass spectrometry (MS/MS) experiments. The structures of major degradation products were identified with MS and specifically labelled nitric acid. Although NPOE and calix[4]arene crowns alone are relatively stable, under simulated conditions resembling the real industrial processes involving radiolysis in the presence of nitric acid, several products resulting from nitration and oxidation were observed.

Journal Article↗

PubMed2003

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry.

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MS(n) experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6beta (1); 6alpha-7alpha (2); 6alpha-6beta-7alpha (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MS(n). The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MS(n) experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones.

Chromatography, Liquid↗

PubMed2003

Irradiation effects in MALDI, ablation, ion production, and surface modifications. Part II. 2,5-dihydroxybenzoic acid monocrystals.

Irradiation effects at low and high laser fluence on 2,5-dihydroxybenzoic acid large crystals were investigated. Contrary to what was observed for matrices as cinnamic acid derivatives, no chemical degradation of matrix is evidenced and continuous ablation as well as ion production resulted of extended irradiation in all the fluence range corresponding to classical matrix-assisted laser desorption /ionization. Ripples are formed on the base of the crater for a limited number of laser shots under moderate fluence. For extended irradiation, conical shape craters are formed with the axis of the crater oriented along the incident direction of the laser beam. A study of the craters showed that ablation through the ablated volume slowly varied with the laser fluence when a strong increase of ion production (matrix and analyte) was recorded. Ablation volume was found to vary non-linearly with the number of laser shots. On a same spot, the ablated volume and the ion production were measured as a function of the laser energy. With an increasing laser energy (or fluence), the ablated volume slowly increases when the ion production strongly increases. This gives evidence of a decoupling between ablation and ionization. Interaction of the plume with the incoming beam is thus probable.

Crystallization↗

PubMed2002

Collision-induced dissociation studies of poly(vinylidene) fluoride telomers in an electrospray-ion trap mass spectrometer.

Although fluorinated polymers are widely used in different applications, they are rarely investigated by "soft ionization" techniques such as matrix-assisted laser desorption/ionization and electrospray ionization (ESI). We report here, the desorption and fragmentation of poly(vinylidene) fluoride (PVDF) telomers in an ion trap mass spectrometer coupled to an ESI source. Protonated and lithiated telomers under collision-induced dissociation resonant excitation show mainly HF losses. Fragmentation of protonated telomers can be rationalized by a double proton-transfer mechanism and that of lithiated and anionic telomers by an ion-dipole mechanism. Both mechanisms predict the formation of a stable aromatic or a linear conjugated species, respectively. For lithiated telomers, we could determine the degree of polymerization (n) from the product ion abundance in MS2 experiments. The nature of the end group plays a substantial role in orienting the fragmentation of the PVDF ions. It is interesting to note that, in MS2 experiments, Li+ and F- act as catalysts in the fragmentation of PVDF telomers. Fragmentation of the PVDF telomer backbone was not observed under any experimental conditions.

Journal Article↗

PubMed2002

Post-source decay time-of-flight study of fragmentation mechanisms of protonated synthetic polymers under matrix-assisted laser desorption/ionization conditions.

Post-source decay (PSD) of three different nylon oligomers desorbed under matrix-assisted laser desorption/ionization (MALDI) conditions was studied and their fragmentation pathways were investigated. The fragmentation of the protonated oligomers is very similar to that of peptides. The b(n)(+), y(n)(+) and z(n)(+) series of ions were observed in abundance in the PSD spectrum. The end groups and the length of the spacer in the repeating unit influence the fragmentation of the different polyamides and the relative abundances of the product ions. Competitive dehydration and deamination reactions were observed, and depend on the nature of the end groups and the repeating units. The PSD spectra are very similar to collision-induced dissociation (CID) spectra obtained under low-energy conditions, implying that the selected precursor ions possess similar average internal energies. All the peaks observed in the PSD spectrum can be rationalized by reasonable fragmentation mechanisms.

Journal Article↗

PubMed2001

Effects of liquid phase composition on salt cluster formation in positive ion mode electrospray mass spectrometry: implications for clustering mechanism in electrospray.

Potassium bromate salt clusters, [KBrO3]nKx(x+), formed by electrospray ionization were studied as a function of solution properties. Clusters with up to 4 positive charges were observed. Their abundance, charge state and distribution were shown to vary with the organic solvent in solution. The effects of 7 solvents, including methanol, ethanol, isopropanol, acetonitrile, acetone, pyridine, and 1,4-dioxane, were thoroughly investigated. Solvents with a low dielectric constant and a high viscosity seem to favor clustering in solution but do not systematically allow high charge state ion formation. On the other hand, cluster charge reduction during desolvation was not correlated with solvent cation affinity over the range of solvents examined. However, ion distribution in mass spectra could be rationalized as a combination of these two competing phenomena. Charge state increases with the cluster size but may be reduced during ion desolvation when high cation affinity solvent molecules are actually involved in the ion solvation shell. This assumption could be envisaged in either Iribarne or Dole mechanisms of ion release in the gas phase. However, intensity profiles of multiply charged clusters could only be understood in terms of the ion evaporation mechanism.

Journal Article↗

PubMed2001

Approach to the determination of insulin-like-growth-factor-I (IGF-I) concentration in plasma by high-performance liquid chromatography-ion trap mass spectrometry: use of a deconvolution algorithm for the quantification of multiprotonated molecules in electrospray ionization.

The insulin-like-growth-factor-I (IGF-I) peptide is known to be a marker for growth hormone administration. The development of a quantification method by electrospray ionization mass spectrometry (ESI-MS) coupled with high-performance liquid chromatography (HPLC) is required. This paper describes a method to quantify IGF-I using the internal standard R3 IGF-I in its oxidized forms. A deconvolution software was used to quantify the set of multi-charged molecules recorded on an ESI ion trap mass spectrometer. The results (i.e., linearity, reproducibility and concentration range) were obtained on standard samples and the described LC-ESI-MS method should be applicable to biological samples.

Algorithms↗

PubMed2000

Sequencing of a branched peptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Chemical degradation methods combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and post-source decay (PSD)-MALDI reflex TOF mass spectrometry (MS) were used to determine the sequence of a peptide branched on to a known peptide backbone. This study was applied to a branched peptide model (derivative of substance P). The branched peptide mimics a digest of a membrane receptor on to which a derivative of substance P was photochemically linked. Chemical degradation based on N-terminal ladder sequencing in combination with MALDI-TOF-MS gave only partial sequence information. Although single PSD mass spectra still remain difficult to interpret unambiguously, PSD-MALDI-TOF-MS was combined with on-target acetylation and H -- D exchange to give a better and successful approach to the unambiguous determination of the complete amino acid side-chain sequence. This study shows the capability of MALDI-TOF-MS to help in characterizing ligand-receptor interactions.

Acetylation↗

PubMed1998

Coupling 2D SDS-PAGE with CNBr cleavage and MALDI-TOFMS: a strategy applied to the identification of proteins induced by a hypochlorous acid stress in Escherichia coli.

A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity. In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible. The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate. Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol.

Bacterial Proteins↗

PubMed1998

Cytogenetic analysis of a parachordoma.

We report the cytogenetic and histopathological findings in a 7-year-old female child with an intranasal tumor that is most consistent with a parachordoma. Karyotypic analysis of the tumor revealed clonal numerical and structural chromosome abnormalities. Seven cells displayed recurrent changes: der(2)t(2;4), del(3q), and the loss of chromosomes 9, 10, 20, and 22. Four cells showed a loss of chromosome 17. To the best of our knowledge, these are the first clonal chromosome abnormalities described in parachordoma.

Child↗

PubMed1998

Different approaches to the identification of a cortisol isomer compound in horse urine.

A threshold concentration for cortisol in equine urine was fixed at 1.0 microgram ml-1 by the racing authorities in 1994. In some circumstances, interlaboratory discrepancies were observed and structural cortisol modification was revealed. In order to elucidate the degradation process and to prevent it, an identification study of the produced compound was carried out. The modified substrate was characterised by the same molecular weight as cortisol and a shorter retention time under the conditions used for the cortisol quantification (M.A. Popot, PhD Thesis, King's College, London, 1996). To identify this isomer, HPLC-APCI-MS and MS-MS methods were applied to the cortisol post-administration extract diluted in the mobile phase which was either a mixture of methanol-water or labelled methanol-water (CH3OD-D2O). Stereochemical effects were studied under these conditions. Deuterium-hydrogen exchange was also monitored by HPLC-APCI-MS. Considering MS and MS-MS data, the hypothesis of isomerisation at C11 giving the 11 alpha-cortisol was rejected. Isotopic labelling has allowed determination of the number of labile hydrogen atoms of the modified cortisol. Cortisol and modified cortisol have the same number of mobile hydrogens. Therefore, the hypothesis of a reduction at C20 along with an oxidation at C11 has also been rejected. Deuterium-hydrogen exchanges could be a useful tool to elucidate the structure of compounds analysed by HPLC-APCI-MS in a complex matrix such as horse urine extract.

Animals↗

PubMed1996

Kinetic analysis of the reaction between d(TTGGCCAA) and [Pt(NH3)3(H2O)]2+ by enzymatic degradation of the products and ESI and MALDI mass spectrometries.

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.

DNA Adducts↗