Tripletof 5600 plus high resolution tandem mass spectrometer

The back-skin tissues from mice treated with IMQ or L-THE were thawed on ice, and metabolites were extracted from 20 µl of each sample using 120 µl of precooled 50% methanol buffer. Pooled quality control (QC) sample were also prepared by combining 10 μl of each extraction mixture. All samples were detected by a TripleTOF 5600 Plus highresolution tandem mass spectrometer (SCIEX, Warrington, United Kingdom) with both positive and negative ion modes. Chromatographic separation was performed using an ultraperformance liquid chromatography (UPLC) system (SCIEX, United Kingdom). The acquired LC-MS data pretreatment was performed using XCMS software.

Frozen feces (100 mg) were thoroughly ground with liquid nitrogen, mixed with 1 mL 50% methanol buffer, and incubated for 10 min. The mixture was stored at − 20 °C overnight to precipitate proteins and then centrifuged at 4000 × g for 20 min. The supernatants were used for liquid chromatography‒mass spectrometry (LC‒MS) analysis to detect metabolites. An ultra-performance liquid chromatography (UPLC) system (SCIEX, UK) equipped with an ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 μm, Waters, UK) was used for chromatographic reversed-phase separation. The TripleTOF 5600 Plus high-resolution tandem mass spectrometer (SCIEX, Warrington, UK) was operated in both positive and negative ion modes to detect metabolites eluted from the column. The TOF mass ranged from 60 to 1200 Da. XCMS software was used for the acquired MS data pretreatments and exported into the mzXML format [36 (link)].

Metabolomics sample collection, preparation, and metabolome profiling were carried out as previously described (Ruiying et al., 2020 (link)). The back skin tissues from mice treated with CRS or control were thawed on ice, and metabolites were extracted from 20 µL of each sample using 120 µL of precooled 50% methanol buffer (methanol and distilled water were mixed in a 1:1 ratio). Then the mixture of metabolites was vortexed for 1 min and incubated for 10 min at room temperature, and stored at −20°C overnight. The mixture was centrifugated at 4,000 g for 20 min, subsequently the supernatant was transferred to 96-well plates. The samples were stored at −80 °C prior to the LC-MS analysis. Pooled quality control (QC) samples were also prepared by combining 10 μL of each extraction mixture. All samples were detected by a Triple TOF 5600 Plus high-resolution tandem mass spectrometer (SCIEX, Warrington, United Kingdom) with both positive and negative ion modes. Chromatographic separation was performed using an ultraperformance liquid chromatography (UPLC) system (SCIEX, United Kingdom). The data acquisition mode was DDA.

The collected samples were thawed on ice, and metabolites were extracted with 50% methanol buffer. Then, all samples were analysed by LC-MS according to the instructions for the system, and a TripleTOF 5600 Plus high-resolution tandem mass spectrometer (SCIEX, UK) was used to detect metabolites eluted from the column. The online KEGG database was used to annotate the metabolites by matching the exact molecular mass data (m/z) of samples with those from a database. We used an in-house fragment spectrum library of metabolites to validate the metabolite identification. Student t-tests were conducted to detect differences in metabolite concentrations between the two phenotypes. The P-value was adjusted for multiple tests using an FDR (false discovery rate). Supervised PLS-DA was conducted to discriminate the different variables between groups. The VIP value was calculated. A VIP cut-off value of 1.0 was used to select important features. For SCFA analysis, phosphoric acid (0.5% v/v) solution was added to the colonic digesta samples, then vortexed for 10 min, ultrasonicated for 5 min and centrifuged at 12,000 r/min and 4 °C for 10 min. Then, the supernatant was collected, and MTBE (containing internal standard) solution was added. After centrifugation at 12,000 r/min for 10 min at 4 °C, the supernatant was aspirated into a sampling bottle for GC–MS/MS analysis.