A 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-adenosyl-methioninamine (dcSAM), homocysteine (Hcy) and methionine (Met), was developed. The method is based on reversed-phase liquid chromatography with positive electrospray ionization (ESI+) 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 0.05 M 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-d(3) as an internal standard, in the case of Met and Ade, Met-(13)C and Ade-(15)N(2) were employed as isotope-labeled 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 (-0.27 +/- 0.68 ppm). The resulting contents of S(C)S(S)-SAM, S(S)-dcSAM, MTA, Ado and Met in the free-cell supernatant of P. aeruginosa was 56.4 +/- 2.1 nM, 32.2 +/- 2.2 nM, 0.91 +/- 0.10 nM, 19.6 +/- 1.2 nM and 1.93 +/- 0.02 mu M (mean +/- SD, n = 4 extractions), respectively. We report also the baseline separation (R(s) >= 1.5) of both diastereoisomeric forms of SAM (S(C)S(S) and S(C)R(S)) and dcSAM (S(S) and R(S)), which can be very useful to establish the relationship between the biologically active versus the inactive species, S(C)S(S)/S(C)R(S) and S(S)/R(S) of SAM and dcSAM, respectively. An additional confirmation of SAM-related metabolites was accomplished by a systematic study of their MS/MS spectra.
Analysis of S-adenosylmethionine and related sulfur metabolites in bacterial isolates of Pseudomonas aeruginosa (BAA-47) by LC-ESI coupled to a hybrid linear quadrupole ion trap (LTQ) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry
CATALDI, Tommaso;
2009-01-01
Abstract
A 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-adenosyl-methioninamine (dcSAM), homocysteine (Hcy) and methionine (Met), was developed. The method is based on reversed-phase liquid chromatography with positive electrospray ionization (ESI+) 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 0.05 M 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-d(3) as an internal standard, in the case of Met and Ade, Met-(13)C and Ade-(15)N(2) were employed as isotope-labeled 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 (-0.27 +/- 0.68 ppm). The resulting contents of S(C)S(S)-SAM, S(S)-dcSAM, MTA, Ado and Met in the free-cell supernatant of P. aeruginosa was 56.4 +/- 2.1 nM, 32.2 +/- 2.2 nM, 0.91 +/- 0.10 nM, 19.6 +/- 1.2 nM and 1.93 +/- 0.02 mu M (mean +/- SD, n = 4 extractions), respectively. We report also the baseline separation (R(s) >= 1.5) of both diastereoisomeric forms of SAM (S(C)S(S) and S(C)R(S)) and dcSAM (S(S) and R(S)), which can be very useful to establish the relationship between the biologically active versus the inactive species, S(C)S(S)/S(C)R(S) and S(S)/R(S) of SAM and dcSAM, respectively. An additional confirmation of SAM-related metabolites was accomplished by a systematic study of their MS/MS spectra.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
