LC–MS metabolomic analysis

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was used to identify exometabolites across samples over time. Metabolites were extracted into ethyl acetate from filtered supernatant samples after acidification with HCl. Both the aqueous and organic phases were dried down, redissolved, and analyzed by LC–MS/MS (Supplementary Note ⁷) using an Agilent 1290 UHPLC system connected to a Thermo Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer equipped with a Heated Electrospray Ionization (HESI-II) source probe. Separation, ionization, fragmentation and data acquisition parameters are specified in Supplementary Data ¹. Briefly, metabolites were separated by gradient elution followed by MS1 and data dependent (top 2 most abundant MS1 ions not previously fragmented in last 7 s) MS2 collection; targeted data analysis was performed by comparison of sample peaks to a library of analytical standards analyzed under the same conditions. Three parameters were compared: matching m/z, retention time and fragmentation spectra using Metabolite Atlas (https://github.com/biorack/metatlas)^111,112. Additional methodological details, including LC–MS parameters and MS resolution, are provided in Supplementary Data ¹. Identification and standard reference comparison details are provided in Supplementary Data ¹. For more information on LC–MS analyses, see Supplementary Note ⁷. To determine significantly discriminating LC–MS exometabolites, we applied a linear model to the log2-transformed peak area data using limma¹¹³ (v3.42.2) in R on log2-transformed data to compare metabolites in live and autoclaved treatments at each timepoint. Limma statistics are given in Supplementary Data ¹.