Progenesis qi for small molecules

Raw data was processed using Xcaliber software version 2.2 (Thermo Fisher Scientific, Waltham, MA, USA) for data obtained from the Q-Exactive and Masshunter for Agilent Data (Agilent Santa Clara, CA, USA). Progenesis QI for small molecules (Waters, Elstree, UK) was used for measuring the number of compound features (compound features had a unique m/z/chromatographic retention time and isotope peaks corresponding to their expected natural abundances). Compound identification and statistical analysis was also performed using Progenesis QI. The Progenesis workflow involved automatic peak alignment, isotope cluster recognition (‘peak picking’), compound identification using a database constructed from the authentic standard analysis (see below) and statistical analysis including PCA analysis. This included the comparison of normalised molecular abundance across samples for identified and unknown compounds. The relationship between peak area and concentration, for a particular compound feature, was used to compare concentrations between samples, when above the limit of quantification (LOQ). The LOQ was determined by ensuring it was above the instrument detection limit where applicable (urolithin A) and a signal to noise ratio of 10:1 (see experimental section below).

Raw data was processed using Progenesis QI for small molecules (Waters, Elstree, UK). Briefly this encompassed chromatographic peak alignment, isotope cluster recognition (peak picking) and compound identification. Identification of compounds in experimental samples was based on matching to an in-house library of authentic standards, using four measured parameters for each compound from the database. These were: accurate mass measurement (<5ppm) based on theoretical mass derived from the chemical formula, experimental retention time window of 0.5mins, isotope pattern recognition (calculated from chemical formula) and matching with fragmentation patterns from an authentic standard where these were available from survey scans. All values in the database were obtained from the analysis of authentic standard compounds.

Data Processing: Raw data was processed using Progenesis QI for small molecules (Waters, Elstree, UK). Briefly, this encompassed chromatographic peak alignment, isotope cluster recognition (peak picking) and compound identification. Identification of compounds in experimental samples was based on matching to an in-house library of authentic standards, using four measured parameters for each compound from the database. These were: accurate mass measurement (<5 ppm) based on theoretical mass derived from the chemical formula, experimental retention time window of 0.5 mins, isotope pattern recognition (calculated from chemical formula) and matching with fragmentation patterns from an authentic standard where these were available from survey scans. All measured values in the database were obtained from the analysis of authentic standard compounds. A full list of identified metabolites and their raw extracted ion chromatogram (EIC) peak areas from each sample can be found in Supplementary Table S4.