Minimal defined medium with 5 g/L glucose

Intracellular metabolites were extracted by incubation in 75% ethanol 10mM ammonium acetate pH 7.5 for 3 min at 95ºC. The supernatant was retained by centrifuging at −9 °C, samples were dried in a vacuum centrifuge and two samples each were prepared for either LC-MS/MS of GC ... -TOF analysis. For quantification by GC-TOF, two sample aliquots were derivatized with first methoxyamine solution (20 mg/ml methoxyamine hydrochloride (Supelco) in pyridine (analytical grade, Merck)) and then either TMS (N-methyl-N-(trimethylsilyl)-trifluoroacetamide (Fluka) or TBDMS N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyl-dimethylchlorosilane (Fluka). The samples were separated via GC on a HP5-MS (Hewlett-Packard, length 30m x ID 0.25 x film 0.25 µm) column and injected (CIS4, Gerstel, Germany) for MS analysis to a TOF spectrometer (Pegasus III, Leco, Germany). Detailed information on process parameters are described in [1] and [2]). Leco ChromaTOF software (version 2.32) was used for machine control. An autosampler (MPS2, Gerstel, Germany, controlled by Gerstel Maestro software, version 1.2.3.5) was used for automatized derivatization and sample injection to the GC-TOF system.
For quantification by LC-MS/MS, an Ion-Pairing LC method adapted from [3] was applied [2]. The mobile phase was composed of eluent A (aqueous solution of 10mM tributylamine and 15mM acetic acid) and eluent B (methanol); the gradient profile was as follows: t=0 min, 0% B; t=15 min, 55% B; t=27 min, 66% B; t=28 min, 100% B. The end-capped C18 column Synergi Hydro RP, 2.1 x 150 mm, 4 µm particles (Phenomenex, Germany) was employed. The column was equilibrated for 20 min before each injection, the flow rate was 200 µl/min and the column temperature was controlled at 40 ºC. For tandem MS analysis a 4000 QTRAP linear ion trap mass Spectrometer (AB Sciex, Canada) was coupled to the LC. Analyst software (AB Sciex, Canada) was used for both machine control and data acquisition. All analyses were performed in negative ion and selected reaction monitoring mode with Q1 and Q3 set to unit resolution. Ion spray voltage, auxiliary gas temperature, nebulizer gas (GS1), auxiliary gas (GS2), curtain gas (CUR) and collision gas (CAD) were set to 4200 V, 650ºC, 65, 40, 10, 4 (arbitrary units), respectively. Nitrogen (Pangas, Switzerland) was used as curtain and collision gas. Declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) were optimized separately for each transition. To obtain temporal resolution of >1 Hz for each transition, the run was divided into five segments and the dwell time for each transition was set to 50ms.

--------------References------------------
[1] Ewald JC, Heux S, Zamboni N (2009). High-throughput quantitative metabolomics: workflow for cultivation, quenching, and analysis of yeast in a multiwell format. Anal Chem 81: 3623–3629. http://doi.org/chgtcq
[2] Buscher JM, Czernik D, Ewald JC, Sauer U, Zamboni N (2009). Cross-platform comparison of methods for quantitative metabolomics of primary metabolism. Anal Chem 81: 2135–2143. http://doi.org/cjqg95
[3] Luo B, Groenke K, Takors R, Wandrey C, Oldiges M (2007). Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147: 153–164. http://doi.org/cxdj7w