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Profiling of N-Acyl-Homoserine Lactones by Liquid Chromatography Coupled with Electrospray Ionization and a Hybrid Quadrupole Linear-Ion-Trap and Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry (LC-ESI-LTQ-FTICR-MS)
No full textA method for the comprehensive profiling of the N-acyl-homoserine lactone (AHL) family of bacterial
quorum-sensing molecules is presented using liquid chromatography (LC) coupled to a hybrid quadrupole
linear ion trap (LTQ) and Fourier-transform ion-cyclotron-resonance mass spectrometer (FTICR). We
demonstrate an increase in signal intensity in MS with electrospray ionization (ESI) of the protonated
molecules, [M + H]+, by using acetonitrile (ACN) instead of methanol (MeOH) as the organic solvent
under the conditions in which the samples were supplied to the probe by direct infusion at constant
flow rates. The presence of ACN prevents the formation of methanol adducts such as [M + MeOH + H]+
and [M + MeOH + Na]+, while also lowering the signal intensity of sodiated [M + Na]+ ions. Sensitivity
of these signaling molecules in terms of signal-to-noise ratio (S/N) using low-resolution LTQ-MS and
high-resolution FTICR-MS were compared under reversed-phase (RP) LC separations with ESI interface.
Special emphasis was paid to the choice of the separation column, its elution conditions and detection of
the major AHL compounds produced by the Serratia liquefaciens strain ATCC 27592. The most promising
results were obtained using a RP C16-amide column eluted with a linear mobile phase gradient ACN/H2O
containing 0.1% formic acid. The whole set of AHL homologs in bacterial extracts was detected in the
extracted-ion chromatographic (XIC) mode, and the calculations of molecular formulae were performed
by including the isotopic pattern. This mode of displaying data, with a very narrow mass-to-charge ratio
window (i.e. ±0.0010 as m/z unit) around each selected ion, has allowed the identification of all the eight
known homoserine lactones, viz. C4-HSL, 3-oxo-C6-HSL, C6-HSL, 3-oxo-C8-HSL, C8-HSL, C10-HSL, C12-
HSL andC14-HSL. In addition, at least four uncommon signalingmediators previously unreported, namely,
3-oxo-C10 : 1-HSL, 3-oxo-C11 : 2-HSL, 3-oxo-C13 : 2-HSL and 3-OH-C16-HSL, were identified and characterized;
their roles in cell-to-cell communication has to be elucidated
Analysis of S-adenosylmethionine and related sulfur metabolites in bacterial isolates of Pseudomonas aeruginosa (BAA-47) by liquid chromatography/electrospray ionization coupled to a LTQ hybrid linear quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry
No full textA comprehensive and highly selective method for detecting in bacterial supernatants a modified sulfur nucleoside, S-adenosyl-L-methionine (SAM), and its metabolites, i.e., S-adenosylhomocysteine (SAH), adenosine (Ado), 5(-deoxy-5(-methylthioadenosine (MTA), adenine (Ade), S-adenosylmethioninamine
(dcSAM), homocysteine (Hcy) and methionine (Met), was developed. The method is based on reversed-phase liquid chromatography with positive electrospray ionization (ESIR)
coupled to a hybrid linear quadrupole ion trap (LTQ) and 7-T Fourier transform ion cyclotron
resonance mass spectrometry (FTICRMS). A gradient elution was employed with a binary solvent of 0.05M ammonium formate at pH 4 and acetonitrile. The assay involves a simultaneous cleanup of cell-free bacterial broths by solid-phase extraction and trace enrichment of metabolites with a 50-fold concentration factor by using immobilized phenylboronic and anion-exchange cartridges. While the quantitative determination of SAM was performed using stable-isotope-labeled SAM-d3 as an internal standard, in the case of Met and Ade, Met-13C and Ade-15N2 were employed as isotopelabeled
internal standards, respectively. This method enabled the identification of SAM and its metabolites in cell-free culture of Pseudomonas aeruginosa grown in Davis minimal broth
(formulation without sulphur organic compounds), with routine sub-ppm mass accuracies
(0.27W0.68 ppm). The resulting contents of SCSS-SAM, SS-dcSAM, MTA, Ado and Met in the
free-cell supernatant of P. aeruginosa was 56.4W2.1 nM, 32.2W2.2 nM, 0.91W0.10 nM, 19.6W1.2nM
and 1.93W0.02mM (meanWSD, n1⁄44 extractions), respectively. We report also the baseline separation (Rs ‡1.5) of both diastereoisomeric forms of SAM (SCSS and SCRS) and dcSAM (SS and RS), which can be very useful to establish the relationship between the biologically active versus the
inactive species, SCSS/SCRS and SS/RS of SAM and dcSAM, respectively. An additional confirmation
of SAM-related metabolites was accomplished by a systematic study of their MS/MS spectra
Improved Determination of Taurine by High-Performance Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection
No full textAs taurine is a very important compound involved in a large number of metabolic processes, it is naturally present in the mammal tissues
and is often deliberately added in some foods as a fortifying component. A detailed knowledge of taurine metabolic roles in biological
systems can be obtained only if a sensitive, reliable and rapid analytical method is available. This article describes the successful application
of high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) for taurine
determination in egg white and yolk samples, as well extracts of human serum and urine. Applications are shown for determination of taurine
in soft drinks and pharmaceutical preparations where the taurine content was evaluated by standard additions. These results were achieved
without prior derivatization of taurine
Scrambling of autoinducing precursor-peptides investigated by infrared multiphoton dissociation with electrospray ionization and Fourier-transform ion cyclotron resonance mass spectrometry
No full textTwo synthetic precursor peptides, H2N-CVGIW and H 2N-LVMCCVGIW, involved in the quorum sensing of Lactobacillus plantarum WCFS1, were characterized by mass spectrometry (MS) with electrospray ionization and 7-T Fourier transform ion cyclotron resonance (ESI-FTICR) instrument. Cell-free bacterial supernatant solutions were analyzed by reversed-phase liquid chromatography with ESI-FTICR MS to verify the occurrence of both pentapeptide and nonapeptide in the bacterial broth. The structural characterization of both protonated peptides was performed by infrared multiphoton dissociation using a continuous CO2 laser source at a wavelength of 10.6 μm. As their fragmentation behavior cannot be directly derived from the primary peptide structure, all anomalous fragments were interpreted as neutral loss of amino acids from the interior of both peptides, i.e., loss of V, G, VG and M, MC, V, CC, from H2N-CVGIW and H 2N-LVMCCVGIW, respectively. Mechanisms of this scrambling are proposed. FTICR MS provides accurate masses of all fragment ions with very low absolute mass errors (<1.6 ppm), which facilitated the reliable assignment of their elemental compositions. The resolving power was more than sufficient to resolve closely isobaric product ions with routine subparts per million mass accuracies. Only the occurrence of pentapeptide was found in the cell-free culture of L. plantarum, grown in Waymouth's medium broth, with a low content of 5.2 ± 2.6 μM by external calibration. Most of it was present as oxidized H2N-CVGIW, that is, the soluble disulfide pentapeptide with a level tenfold higher (i.e., 50 ± 4 μM, n = 3)
Modificazione di analoghi del blu di prussia con rutenio: caratterizzazione elettrochimica e spettroscopica
No full textQuantitative Determination of taurine in real samples by high-performance anion-exchange Chromatography with integrated Pulsed Amperometric Detection
No full textAs taurine is a very important compound involved in a large number of metabolic processes, it is naturally present in the mammal tissues
and is often deliberately added in some foods as a fortifying component. A detailed knowledge of taurine metabolic roles in biological
systems can be obtained only if a sensitive, reliable and rapid analytical method is available. This article describes the successful application
of high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) for taurine
determination in egg white and yolk samples, as well extracts of human serum and urine. Applications are shown for determination of taurine
in soft drinks and pharmaceutical preparations where the taurine content was evaluated by standard additions. These results were achieved
without prior derivatization of taurine
Determination Of Mono- And Disaccharides In Milk And Milk Products By High-Performance Anion-Exchange Chromatography With Pulsed Amperometric Detection
No full textA simple and sensitive liquid chromatographic method for the separation and quantification of mono- and disaccharides
in raw- and processed-milk is described. Samples of cows’, buffalos’, sheeps’ and goats’ milk were analyzed upon clarification
and appropriate dilution for the quantification of lactose, galactose, glucose and N-acetylglucosamine (GlcNAc).
The separation was accomplished by high-performance anion-exchange chromatography with pulsed amperometric detection
(HPAEC–PAD), using a gold working electrode and dilute alkaline eluents modified by a millimolar concentration of barium
acetate. The eluent composition employed was designed to provide optimum separation with respect to the selected sample,
without interference from the matrix components. The analytical method was successfully employed for the determination of
mono- and disaccharides naturally occurring in dairy milk, mozzarella cheese and whey samples, with high sensitivity and
accuracy
Determinazione della taurina in matrici alimentari mediante cromatografia a scambio anionico e rivelazione amperometrica pulsata
No full textFragmentation pathways of synthetic peptides investigated by infrared multiphoton dissociation (IRMPD) and Fourier-transform ion cyclotron mass spectrometry (FTICR MS)
No full textAccurate mass analysis of N-acyl-homoserine-lactones and cognate lactone-opened compounds in bacterial isolates of Pseudomonas aeruginosa PAO1 by LC-ESI-LTQ-FTICR-MS
No full textN-acyl-homoserine-lactones (AHSLs) are widely conserved signal molecules present in quorum sensing systems of Gramnegative
bacteria such as Pseudomonas aeruginosa. We present here the results obtained with a hybrid linear trap/Fourier
transform ion cyclotron resonance (LTQ-FTICR) mass spectrometer used to investigate the occurrence of AHSLs and cognate
N-acyl-homoserines (AHSs) in bacterial isolates of P. aeruginosa (strain PAO1). Two hydrolysed AHSs were found in significant
amounts, most likely formed through the lactone opening of N-3-oxo-decanoyl-L-homoserine-lactone (3OC10-HSL) and
N-3-oxo-dodecanoyl-L-homoserine-lactone (3OC12-HSL). Structure elucidation of these ring-opened molecules, i.e. N-3-oxodecanoyl-
L-homoserine (3OC10-HS), and N-3-oxo-dodecanoyl-L-homoserine (3OC12-HS), which are not detected by bacterial
biosensors, was performed by high-resolution and accurate mass measurements upon liquid chromatography (LC) and
confirmed by tandem MS in the LTQ analyser. Assignment of chemical formula, with mass spectra in the form of [M + H]+, was
significantly expedited by extracted ion chromatograms (XICs) because the number of potentially plausible formulae for each
protonated signalling molecule was considerably reduced a priori by the LC behaviour, the high mass measurement accuracy
available in FTICR mass spectra and the isotopic patterns. At least two concentration levels were observed in spent culture
supernatants of P. aeruginosa: compounds at a relatively high content (5–15 μM) that is C4-HSL, 3OC10-HS, and 3OC12-HS and
those occurring at a lower content (<0.2 μM) that is C6-HSL and C8-HSL. The implications of this work extend to a great variety
of Gram-negative bacteria
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