Liquid chromatography-tandem quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry measurement of targeted metabolites of Methylobacterium extorquens AM1 grown on two different carbon sources.
Complementary methods using liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOF-MS) were developed and applied to determine targeted metabolites involved in central carbon metabolism [including tricarboxylic acid cycle, serine cycle, ethylmalonyl-coenzyme A (ethylmalonyl-CoA) pathway and poly-beta-hydroxybutyrate cycle] of the bacterium Methylobacterium extorquens AM1 grown on two carbon sources, ethylamine (C2) and succinate (C4). Nucleotides, acyl-CoAs and a few volatile metabolites in cell extracts of M. extorquens AM1 were readily separated using either hydrophilic interaction liquid chromatography or reversed-phase liquid chromatography, and detected with good sensitivity by MS/MS. However, volatile intermediates within a low mass range (<300 m/z), especially at low abundance (such as glyoxylic acid and others <500nM), were more effectively analyzed by GCxGC-TOF-MS which often provided better sensitivity, resolution and reproducibility. The complementary nature of the LC-based and GC-based methods allowed the comparison of 39 metabolite concentrations (the lowest level was at 139.3nM). The overlap between the LC-based and GC-based methods of seven metabolites provided a basis to check for consistency between the two methods, and thus provided some validation of the quantification accuracy. The abundance change of 20 intermediates further suggested differences in pathways linked to C2 and C4 metabolism.
Yang
Song
S
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2180, USA.
Sadilek
Martin
M
Synovec
Robert E
RE
Lidstrom
Mary E
ME
D002244:Carbon / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D003627:Data Interpretation, Statistical; D005021:Ethylamines / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods; D020601:Methylobacterium extorquens / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019802:Succinic Acid / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D005021:Ethylamines; D002244:Carbon; D019802:Succinic Acid; C041564:ethylamine