Fecal metabolites analysis
SEPARATION
Metabolomics in feces were analyzed at Shanghai Personal Biotechnology Cp. Ltd using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600). Stool weighing 100 mg was added with 400 ml extract of methanol-acetonitrile-water (2:2:1, vol/vol/vol), vortexed and centrifuged at 14,000 g for 20min, and the supernatant was collected for further measurement. For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 µm column (waters, Ireland). In both ESI positive and negative modes, the mobile phase contained A=25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. For RPLC separation, a 2.1 mm × 100 mm ACQUIY UPLC HSS T3 1.8 µm column (waters, Ireland) was used. In ESI positive mode, the mobile phase contained A= water with 0.1% formic acid and B= acetonitrile with 0.1% formic acid; and in ESI negative mode, the mobile phase contained A=0.5 mM ammonium fluoride in water and B= acetonitrile. The raw MS data (wiff.scan files) were converted to MzXML files using ProteoWizard MSConvert before imported into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 10 ppm, peak width = c (10, 60), prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was used for annotation of isotopes and adducts.After normalized to total peak intensity, the processed data were analyzed by R package, where it was subjected to multivariate data analysis, including Pareto-scaled principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). The 7-fold cross-validation and response permutation testing were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Metabolites with the VIP value > 1 were further applied to Student’s t-test at the univariate level to measure the significance of each metabolite. The 