Metabolomics data from A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle

Abstract

Metabolomics data was prepared as following. Freezer stocks of wt (BW25113) and ΔycgM E. coli were streaked on LB plates and 4 single colonies from each strain were grown for 24 h in M9 minimal medium (0.2% glucose). Cells were washed with 1x M9 salts and used to inoculate 5 mL culture tubes containing M9 medium with either 0.2% glucose (natural isotopic abundance) or 0.2% 13C6-glucose (fully labeled) to an initial OD600 = 0.05. Four replicate cultures of each genotype were grown in each media formulation by shaking at 37°C for ~4 h until cells reached mid-log growth phase (OD600 ~0.6; determined with a Thermo Scientific Genesys 30 spectrophotometer that can measure OD inside culture tubes). To minimize metabolic disturbances, a rapid harvesting protocol was used. Briefly, an equivalent of 1 mL at OD600 = 1.0 was quickly transferred to 1.5 mL polypropylene tubes, cells were pelleted in a microcentrifuge at full speed for 30 s, the supernatant was quickly aspirated and collection tubes were immediately snap frozen in liquid nitrogen. Samples were stored at -80°C prior to extraction. After collecting the mid-log samples, cultures were shaken for an additional 2 h until early stationary growth phase (OD600 ~1.2) and samples were harvested as above. Collection tubes were placed on dry ice, 0.2 mL of cold 90% methanol was added, and tubes were incubated at -80°C for 72 h. Samples were removed from the freezer, vortexed for 15 s, and incubated on ice for 3 h with vortexing every ~30 min. Afterwards samples were spun for 15 min in a microcentrifuge (16,000 g) at 4°C and supernatants were collected for analysis. Metabolomic data were obtained using an ultra-performance liquid chromatography-electrospray ionization-hybrid quadrupole-orbitrap mass spectrometer (Ultimate® 3000 HPLC, Q Exactive™, Thermo Scientific) with an autosampler and with a sample vial block maintained at 4°C. Chromatographic separations were carried out on an SeQuant® ZIC®-cHILIC 3µm, 100Å, 100 x 2.1 mm column (Merck, Darmstadt, Germany) with column temperature 40°C, flow rate 0.40 mL/min, and a 2 μL injection volume. Mobile phases A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile were delivered over a 23 min. gradient according to the following profile: initial 98% B, 2 min 98% B, 20 min 40% B, 22 min 98% B, 23 min 98% B. The MS conditions used were full scan (mass range 50-750 m/z, and 115-1000 m/z in separate analyses), resolution 70,000, desolvation temperature 350°C, spray voltage 3800 V, auxiliary gas flow rate 20, sheath gas flow rate 50, sweep gas flow rate 1, S-Lens RF level 50, and auxiliary gas heater temperature 300°C. Xcalibur™ software version 2.1 (Thermo Scientific) was used for data collection. Tandem MS data were obtained using data dependent Top N acquisition (Full MS & dd-MS/MS). Precursor ions (top 5 most abundant ions per scan) were sequentially fragmented in the HCD collision cell with normalized collision energies (NCE) of 10, 20, 30, 40, 50, and 60 for six independent injections of each sample. MS/MS scans were acquired with 17,500 resolution, target value 1.0 × 105, 50 ms maximum injection time, and isolation window of 4.0 m/z. Data files were converted from .RAW to .mzML and .mgf formats using the ProteoWizard tool MSConvertGUI. MZmine 2.53 was utilized for extracting exact-mass chromatographic data for isotope ratio calculations, for generating untargeted metabolite feature tables, and for tabulating peak heights from targeted exact masses for calculating relative signal intensity ratios.

Key for sample names: 1st position letter: A = wild-type, B = OAT1 KO (ΔycgM) 2nd position number: 2 = natural glucose, 3 = 13C-glucose 3rd position letter: M = mid-log phase, S = early stationary phase 4th position number: 1-4 = biological replicates (mid-log and early stationary samples are paired; i.e., A2M1 and A2S1 were collected from the same culture tube)

example: A2M1 = wild type E. coli grown with natural glucose and harvested at mid-log, replicate 1