Untargeted In Vitro Metabolomics data from Burkholderia cenocepacia J2315, H111 and Staphylococcus aureus NRS77 Biofilm Supernatant

Here, we offer a metabolomics dataset generated via high-performance-liquid chromatography and high-resolution mass spectrometry analysis of in vitro biofilm supernatant harvested from the human pathogens Burkholderia cenocepacia H111 & J2315 and Staphylococcus aureus NRS77. Polar metabolites from uninoculated media (LB+1% glucose, 150 mM MOPS) is also included as a control condition.METHODSSample CollectionB. cenocepacia or Staphylococcus strains were inoculated into T LB Lennox + 1% glucose + 150 mM MOPS at 10^6 CFU/mL and incubated at 37 °C for 3 days in replicate wells of a 96-well PVC plate. After 7 days, the biofilm supernatants were removed via a micropipette, centrifuged at 21,130 x g for 1 min, and filter-sterilized through a 0.45 µm syringe filter (VWR 28145-505).ExtractionSterile supernatant was collected for each sample and frozen at -80 °C for 2 h or overnight, then freeze dried in a LabConco 2.5L Benchtop Freeze Dryer. Dried sample was reconstituted into 2 mL 50% acetonitrile and transferred into an autosampler injection vial after filtered with a 0.22 µm filter.ChromatographyAll samples were run on a Thermo DIONEX UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The LC system was equipped with a reversed phase column (RPC, a Waters Acquity UPLC HSS T3 column, 2.1 x 150 mm, 1.8 µm) and hydrophilic interaction chromatography column (HILIC, a Millipore SeQuant ZIC-cHILIC column, 2.1 x 150 mm, 3 µm). The two LC columns were configured in parallel, and each column was connected with a 2-μL sample loop. The column temperature was set to 40 °C. For separation on the RPC column, water with 0.1% formic acid and acetonitrile with 0.1% formic acid were mobile phase A and B, respectively. The flow rate was 0.35 mL/min. The solvent gradient was set as 0-6 min 0% B, 6-14 min increased from 0% to 28% B, 14-16 min increased from 28% to 50% B, 16-20 min increased from 50% to 100% B, 21-33 min back to 0% B. For separation on the HILIC column, 10 mM ammonium acetate (pH=3.25) and acetonitrile with 0.1% formic acid were mobile phase A and B, respectively. The flow rate was 0.3 mL/min. The solvent gradient was set to 0-1.3 min 95% B, 1.3-8.3 min decreased from 95% to 0% B, 8.3-11 min kept 0% B, 11.5-33 min kept 95% BMass SpectrometryAll samples were run on a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. All samples were randomly analyzed in positive (+) and negative (-) modes to obtain full MS data for metabolite quantification. For the metabolite identification, the pooled sample was analyzed by 2D LC-MS/MS in positive and negative modes at three collision energies, 20, 40 and 60 eV.Data TransformationThe raw data was converted mzML format using the MSCovert software, and XCMS software was used for spectrum deconvolution[1] and MetSign software for metabolite identification, cross-sample peak list alignment, normalization and statistical analysis[2][3].Refs:[1] Tautenhahn, R., Patti, G.J., Rinehart, D., and Siuzdak, G. (2012). XCMS On-line: a web-based platform to process untargeted metabolomic data. Anal Chem 84, 5035-5039. 10.1021/ac300698c.[2] Wei, X., Shi, X., Kim, S., Patrick, J.S., Binkley, J., Kong, M., McClain, C., and Zhang, X. (2014). Data dependent peak model based spectrum deconvolution for analysis of high resolution LC-MS data. Anal Chem 86, 2156-2165. 10.1021/ac403803a.[3] He, L., Li, F., Yin, X., Bohman, P., Kim, S., McClain, C.J., Feng, W., and Zhang, X. (2019). Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease. J Proteome Res 18, 2875-2884. 10.1021/acs.jproteome.9b00181.Metabolite IdentificationTo identify metabolites, 2D LC-MS/MS data was first matched to our own proprietary database that contains parent ion m/z, MS/MS spectra and retention time of 363 authentic standards. Thresholds were set as spectral similarity ≥ 0.4, retention time difference ≤ 0.15 and m/z variation window ≤ 5 ppm. 2D LC-MS/MS data without a match with the metabolites in the proprietary database were further analyzed using Compound Discoverer software (v 2.0, Thermo Fisher Scientific, Germany), where MS/MS spectra similarity score threshold was set ≥ 40 with a maximum score of 100. The suite of databases utilized though Compound Discoverer v3.1 software are as follows: E. coli Metabolome Database; Fecal Metabolome database; KEGG; Saliva Metabolome Database; Urine Metabolome Database; Compound Classes/Therapeutics/Prescription Drugs (including only the Endogenous Metabolites; Natural Products/Medicines; Natural Toxins; Small Molecule Chemicals; Steroids/Vitamins/Hormones; and Therapeutics/Prescription Drugs groups).