MassIVE MSV000090542

Description

Samples for metabolomic analysis were prepared as follows. A 200 uL aliquot of bacterial cultures or control medium (prepared, as described in the above section) was mixed with 800 uL of 99.5 percent ethanol (Fisher Scientific) and subjected to 3 rounds of freeze thaw cycles: 10 minutes at -80C, 8 minutes at room temperature. Next, samples were spun down for 10 minutes at 16,000g, 4C to remove cell debris, and supernatants were transferred to new tubes. Ethanol supernatants were stored at -80C until further extraction. Each ethanol extraction was performed in two technical duplicates. The solid phase extraction (SPE) of the ethanol extracts was performed with the Oasis HLB 96-well plate (30 mg Sorbent per Well, 30 um Particle Size) (Waters Corporation) set up with a vacuum pump manifold. Each well of the plate was washed with 700 uL of 100 percent HPLC-grade methanol and equilibrated with 700 uL of HPLC-grade water. Next, 400 uL of ethanol extracts were added to the wells and allowed to slowly elute. Wells were next washed with 800 uL of 5 percent methanol in water. Metabolites from bacterial cultures or control media were eluted with 200 uL of 100 percent methanol. Vacuum up to 20 psi was applied for the wells that did not elute within one hour. The collected eluted metabolites were stored at -20 until the HPLC-MS analysis. The HPLC-MS analysis of extracted metabolites was performed with the use of a Dionex UltiMate 3000 UHPLC system (Fisher Scientific) coupled to a Bruker impact HD quadrupole time-of-flight mass spectrometer (Bruker). The chromatographic separation was performed on a Kinetex C18 1.7 um, 100A UHPLC column (50 mm x 2.1 mm) (Phenomenex), held at 40C during analysis. 5 uL of each sample was injected. Mobile phase A was water, 0.1 percent v:v formic acid, and mobile phase B was acetonitrile, 0.1 percent v:v formic acid. The solvent gradient table was set as follows: 2 percent B, increased to 10 percent B over 0.2 min, then to 100 percent B at 12 min, held at 100 percent B for 1.5 min, decreased back to 2 percent B in 0.5 min, followed by washout cycle and equilibration; total analysis time of 15 min. The scanned m/z range was 80-2000 Th; capillary voltage 4,500 V; nebulizer gas pressure 2 bar, drying gas flow rate 9 l min-1 and temperature 200C. The full MS scan was followed by a tandem mass spectrometry fragmentation of the seven most abundant ions within the spectrum. For tandem mass spectrometry, the collision cell collision energy was set at 3 eV and the collision energy was stepped 50 percent, 75 percent, 150 percent, and 200 percent. The scan rate was 3 Hz. A HP-921 lock mass compound was infused during the analysis for post processing mass correction. [doi:10.25345/C50000485] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Cellular Homeostasis ; Genetic Modification ; Genome Editing ; microbial culture ; gene deletion ; Metabolomics

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