Analysis of S-adenosylmethionine and related sulfur metabolites in bacterial isolates of Pseudomonas aeruginosa (BAA-47) by liquid chromatography/ electrospray ionization coupled to a hybrid linear quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry

摘要

A comprehensive and highly selective method for detecting in bacterial supernatants a modifiedsulfur 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 methodis 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 cyclotronresonance mass spectrometry (FTICRMS). A gradient elution was employed with a binary solvent of0.05 M ammonium formate at pH 4 and acetonitrile. The assay involves a simultaneous cleanup ofcell-free bacterial broths by solid-phase extraction and trace enrichment of metabolites with a 50-foldconcentration factor by using immobilized phenylboronic and anion-exchange cartridges. While thequantitative determination of SAM was performed using stable-isotope-labeled SAM-d3 as aninternal standard, in the case of Met and Ade, Met-~(13)C and Ade-~(15)N_2were employed as isotope-labeled internal standards, respectively. This method enabled the identification of SAM andits 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_CS_S-SAM, S_S-dcSAM, MTA, Ado and Met in thefree-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 nMand 1.93 ± 0.02 (mean ± SD, n= 4 extractions), respectively. We report also the baseline separ-ation (Rs ≥1.5) of both diastereoisomeric forms of SAM (S_CS_S andS_C_R_S)and dcSAM (S_Sand S),which can be very useful to establish the relationship between the biologically active versus theinactive species, S_CS_S/S_CR_S and S_S/R_S of SAM and dcSAM, respectively. An additional confirmationof SAM-related metabolites was accomplished by a systematic study of their MS/MS spectra.