Quantitative liquid chromatography-tandem mass spectrometry profiling of activated methyl cycle metabolites involved in LuxS-dependent quorum sensing in Escherichia coli.
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 MG1655 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 [(13)CD(3)]methionine and S-adenosylcysteine. Linearity of the assay was established up to a concentration of 700 microg/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 Deltapfs and DeltaluxS mutants to determine the metabolic consequences of a disruption to the activated methyl cycle and, hence, LuxS-dependent quorum sensing.
Halliday
N M
NM
School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Hardie
K R
KR
Williams
P
P
Winzer
K
K
Barrett
D A
DA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009154:Mutation; D053038:Quorum Sensing; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria