期刊
ANALYTICAL BIOCHEMISTRY
卷 403, 期 1-2, 页码 20-29出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.04.021
关键词
Metabolite profiling; Quorum sensing; Activated methyl cycle; LuxS; Pfs; LC-MS/MS; Alkyl chloroformates; Escherichia coli
资金
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/D007038/1]
- Biotechnology and Biological Sciences Research Council [BB/D007038/1] Funding Source: researchfish
A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry assay has been developed and validated for the simultaneous quantification of the metabolites and precursors of the activated methyl cycle, reported in preliminary form by Heurlier et al. (2009) [43]. Analytes were extracted from Escherichia coli MG 1655 and chemically derivatized as N(O,S)-iso-butyloxycarbonyl iso-butyl esters using iso-butyl chloroformate in an aqueous iso-butanol/pyridine environment. S-Adenosylmethionine, S-adenosylhomocysteine, S-ribosylhomocysteine, homocysteine, methionine, cystathionine, cysteine, and homoserine were quantified by liquid chromatography-positive ion tandem electrospray ionization mass spectrometry. Internal standards were isotopically labeled [(CD3)-C-13]methionine and S-adenosylcysteine. Linearity of the assay was established up to a concentration of 700 mu g/g cell dry weight for each analyte. The validated assay was used to quantitatively profile the intracellular activated methyl cycle metabolites as a function of growth in E. coli MG1655 and its derivative Delta pfs and Delta luxS mutants to determine the metabolic consequences of a disruption to the activated methyl cycle and, hence, LuxS-dependent quorum sensing. (C) 2010 Elsevier Inc. All rights reserved.
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