(SSBM-P) medium for studying the effect of phosphate depletion: Na-glutamate, 55.2 g/L; D-glucose, 40 g/L; MgSO4, 2.0 mM; phosphate, 4.6 mM; supplemented minimal medium trace element solution SMM-TE [39], 8 mL/L and TMS1, 5.6 mL/L. TMS1 consisted of FeSO4 × 7 H2O, 5 g/L; CuSO4 × 5 H2O, 390 mg/L; ZnSO4 × 7 H2O, 440 mg/L; MnSO4 × H2O, 150 mg/L; Na2MoO4 × 2 H2O, 10 mg/L; CoCl2 × 6 H2O, 20 mg/L, and HCl, 50 ml/L. (SSBM-E) medium for studying the effect of L-glutamate depletion: identical to SSBM-P except for the concentrations of Na-glutamate and phosphate adjusted to be 15 g/L and 9.2 mM, respectively.

Metabolite Extraction - samples stored at −80 °C were completely thawed on an ethanol bath at −23 °C. An internal standard mix was added to each 25 mL sample (biomass from 5 mL sample on filter in 25 mL 60% methanol solution) yielding final concentrations of 3.34 mM D3-alani ... ne, 312.5 μM D4-succinate, 1.67 μM D8-valine, 62.5 μM 13C6-glucose, 0.416 μM 13C10, 15N5-adenosine monophosphate and 1.04 μM 13C1-α-ketoisocaproic acid). This standard mix included compounds to be used as internal standards in different analytical methods for metabolites with different chemical properties (organic acids, phosphometabolites, sugars). Samples were then subjected to three cycles of freezing on liquid nitrogen and thawing at −23 °C on the ethanol bath, found to be sufficient for reaching a maximum of compound extraction into the 60% methanol, and thereafter centrifuged for 5 min at −9 °C and 6000 × g. Supernatants were transferred to new tubes pre-chilled at −23 °C and then divided into aliquots á 6 mL in 15 mL screw cap tubes for analysis using different metabolite profiling methods. Samples were frozen at −80 °C and subsequently subjected to solvent evaporation on a freeze-dryer for 24 h. The freeze-dried samples were stored at −80 °C until MS analysis.
Metabolite Derivatization with Methyl Chloroformate (MCF) and GC-MS Analysis - dried extract samples were dissolved in a solvent mixture consisting of 380 μL 1 M NaOH, 333 μL 100% MeOH, and 67 μL pyridine following a modified protocol of Villas-Boas et al. [1]. 20 μL 1 mM D5-glutamate was added to each sample as an analytical internal standard, and the dissolved sample was then transferred to a silanized 5 mL glass tube. While vortex mixing, the following steps were performed for derivatization with MCF, extraction with chloroform, and stopping the reaction with sodium hydrogencarbonate: 40 μL MCF added, 30 s vortex mixing, 40 μL MCF added, 30 s vortex mixing, 400 μL chloroform added, 10 s vortex mixing, 400 μL 50 mM NaHCO3 added, 10 s vortex mixing. The chloroform phase was dried with anhydrous Na2SO4 prior to GC-MS analysis. In general, 12 to a maximum of 24 samples were derivatized and subsequently analyzed in one sequence. In addition to the samples to be analyzed, each sequence contained several control runs including blank, chloroform and derivatized amino acid and organic acid standard mix samples before and after the biological samples to detect and potentially correct for instrumental variation during the sample series. GC-MS was performed using an Agilent 7890GC-5975MS system, EI source operated at 70 eV, equipped with a 30 m × 250 μm × 0.25 μm Agilent 122-5532G DB-5MS+DG capillary column. The data acquisition method was run in constant pressure mode with an operating pressure of 1 bar. D5-glutamate was used for retention time locking, which enabled retention time correction as columns were cut during maintenance operations. 2 μL sample was injected in pulsed split-less mode, and the metabolites were separated by using a 10 °C/min temperature gradient from 45 °C to 300 °C. The MS was operated in scan mode (start after 6 min, mass range 50–550 a.m.u. at 2.5 scans/s). The GC-MS data were analyzed semi-automatically using the Agilent ChemStation DRS (Deconvolution Reporting Software) and the AMDIS (NIST) deconvolution software using an in-house DRS library containing fifty metabolites. After automatic peak identification and integration, all compound peaks were inspected visually for the correct peak selection (retention time, qualifier ions) and the consistent peak integration, and manual correction was performed if necessary. To further assess the resulting dataset, the average, standard deviation, minima and maxima in retention time for respective compound peaks found in the 32 to 36 GC-MS runs (one time-series distributed over up to three sequences) were calculated. By that means, potential errors concerning peak choice were identified and corrected.
LC-MS/MS Analysis - LC-MS/MS analysis was based on the method introduced by Luo and co-workers [2] and performed on an Agilent 1200 series LC connected via an electrospray ion source to an Agilent 6410 triple quadrupole MS instrument. Forty-two common phosphorous containing metabolites were included in this MS/MS method, and collision energies were optimized for each individual metabolite. For the LC-MS/MS analysis, sequence variability was evaluated by quantification of the internal standards added to the samples prior to metabolite extraction.

----------------References---------------
[1] Villas-Boas, S.G.; Delicado, D.G.; Akesson, M.; Nielsen, J. Simultaneous analysis of amino and nonamino organic acids as methyl chloroformate derivatives using gas chromatography-mass spectrometry. Anal. Biochem. 2003, 322, 134–138. http://doi.org/fph2m9
[2] Luo, B.; Groenke, K.; Takors, R.; Wandrey, C.; Oldiges, M. Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J. Chromatogr. A 2007, 1147, 153–164. http://doi.org/cxdj7w