Masshunter qualitative analysis software

Frozen stool samples (~1 mg) from FAP or UC pouch were inoculated into a 5 mL of mega medium supplemented with 100 M cholic acid-2,2,4,4-d₄ (d₄-CA, Sigma-614149) and chenodeoxycholic acid-2,2,4,4-d₄ (d₄-CDCA, Sigma- 614122) as described in the literature (Goodman et al., 2011 (link)). After incubation under strict anaerobic conditions in a Coy chamber at 37 °C for 2 days, 1 mL of aliquots were taken an d frozen at −80 °C until use. To extract the metabolites, 100 μL of bacterial cultures were mixed with 400 μL of methanol, and then centrifuged at 13,000g for 10 min at 4 °C. The cell-free supernata nts were subsequently filtered through a Durapore PVDF 0.22-μm membrane using Ultrafree centrifugal filters (Millipore, UFC30GV00), and 5 μL of the filtrate was injected into LC-MS. The compounds were separated and analyzed using Agilent UPLC Q-TOF platform as described in the literature (Studer et al., 2016 (link)). The MassHunter Qualitative Analysis Software (Agilent, version 7.0) was used for targeted feature quantification, including standard parameters.

Raw data files were processed using Agilent MassHunter Qualitative Analysis Software (B346). Molecular feature extraction (MFE) generates information in features (compounds) that are stored in files for downstream comparative data analysis by MassProfiler Professional Software (Agilent). The analytical approach employs an algorithm for data alignment across multiple groups and treats all imported files as a single dataset. Following feature alignment and quality control, the unsupervised pattern recognition algorithm principal component analysis (PCA) was chosen to examine data sets for similarly changing features, expected and unexpected clusters, and the presence of outlying samples. Compounds determined to be differentially expressed based on statistical criteria and PCA results were assigned tentative formulae by utilizing a molecular formula generator (MFG) algorithm in Massprofiler Professional or MassHunter Qualitative Analysis. The MFG algorithm assigned molecular formula based on an optimized goodness-of-fit, considering accurate measurements of mass defect, natural isotopic abundance rations, and spacing between isotope peaks. A putative compound ID was tentatively assigned based on the MFG score and confirmed by comparison with retention times and MS/MS fragmentation of pure chemical standards.

Suspect screening for GC-EI-QTOF-MS employed Agilent MassHunter Qualitative Analysis software using a Find by Formula workflow similar to the LC-QTOF-MS workflow. The Agilent GC/Q-TOF – Pesticide PCDL containing 750 pesticides with exact mass fragments and retention times was used (Fig. 1). In contrast to the LC-QTOF-MS workflow, the molecular ion was set as optional, a retention time tolerance of ± 0.2 min was included and the minimum number of qualified fragments was two (see SI-5.2). After manual inspection of the automatically detected compounds, reference standards were purchased for complete identification and for retrospective quantification.