Pegasus 4 time of flight mass spectrometer

Metabolome analysis of the fecal and serum samples was performed at the West Coast Metabolomics Center (University of California, Davis, CA, USA) using a gas chromatography–time-of-flight mass spectrometry (GC-TOF MS) method. Samples were extracted using degassed acetonitrile. Internal standards, C08-C30 fatty acid methyl ethers (FAMEs), were added to the extracts. Samples were subsequently derivatized using methoxyamine hydrochloride in pyridine and then with N-methyl-N-trimethylsilyltrifluoroacetamide for the trimethylsilylation of acidic protons. Analytes were separated using an Agilent 6890 gas chromatograph (Santa Clara, CA, USA), and mass spectrometry was executed on a Leco Pegasus IV time-of-flight mass spectrometer (St. Joseph, MI, USA) according to a previously published protocol [31 (link)]. Unnamed peaks were excluded from statistical analysis, and peak height data were uploaded to MetaboAnalyst 4.0 (Xia Lab, McGill University, Montreal, QC, Canada). Finally, the filtered data were normalized using log transformation and Pareto scaling.

Serum samples were analyzed at the West Coast Metabolomics Center (University of California, Davis, CA, USA) using a gas chromatography–time-of-flight mass spectrometry (GC-TOF MS) method. Serum aliquots were extracted with degassed acetonitrile. Internal standards C08-C30 fatty acid methyl ethers (FAMEs) were added, and the samples were derivatized with methoxyamine hydrochloride in pyridine and subsequently with N-methyl-N-trimethylsilyltrifluoroacetamide for the trimethylsilylation of acidic protons. Analytes were separated using an Agilent 6890 gas chromatograph (Santa Clara, CA, USA), and mass spectrometry was performed on a Leco Pegasus IV time-of-flight mass spectrometer (St. Joseph, MI, USA), following a published protocol [44 (link)]. Unnamed peaks were excluded from statistical analysis and peak height data were obtained and uploaded to MetaboAnalyst 4.0 (Xia Lab, McGill University, Montreal, Canada). Then, the filtered data were normalized through log transformation and Pareto scaling [45 (link),46 (link)].

Methods for metabolomics analysis were described previously (5 (link)). Briefly, plasma and urine samples were submitted for untargeted metabolomics analysis to the West Coast Metabolomics Center at UC Davis. Untargeted metabolomics profiling was performed using an Agilent 7890A gas chromatograph and a Leco Pegasus IV time-of-flight mass spectrometer. BinBase database was used to identify metabolites by retention index, align mass spectra, and perform gap filling. Data was reported as mass spectral peak height.

A Leco Pegasus IV time of flight mass spectrometer was controlled by the Leco ChromaTOF software vs. 2.32 (St. Joseph, MI). The transfer line temperature between gas chromatograph and mass spectrometer was set to 280°C. Electron impact ionization at -70 eV was employed with an ion source temperature of 250°C. Acquisition rate was 17 spectra/second, with a scan mass range of 85 to 500 Da. The column was a Restek corporation Rtx-5Sil MS (30m x 0.25 mm I.D. with 0.25 μm 95% dimethyl/5% diphenylpolysiloxane film). The mobile phase was helium, column temperature 50 to 330°C, flow rate 1 ml/min. Injection volume was 0.5 ml, injection temperature 50°C ramped to 250°C at 12°C/s. Oven temperature program was 50°C 1 min, then ramped at 20°C/min to 330°C, and finally held for 5 min.

Metabolomic data were acquired at the UC Davis West Coast Metabolomics Center. For each sample, approximately 300 absorbance at 600 nm unit (for QA) cells grown to late-logarithmic phase in SC (∼16 h growth from an absorbance of 0.1 at 600 nm) were collected by centrifugation. Snap freezing was achieved by dry ice. Frozen cell pellets were kept in Eppendorf tubes and then subject to metabolite extraction and MS analysis. Cells were extracted following recommendations published before (77 (link)). GC-TOF was performed with an Agilent 6890 gas chromatography instrument with an Rtx-5Sil MS column coupled to a Leco Pegasus IV time of flight mass spectrometer (78 (link)). For data processing, ChromaTOF version 4.50.8 was used in conjunction with the BinBase algorithm as previously described (79 (link)). Metabolite identifications were performed according to the Metabolomics Standards Initiative by using chromatography-specific databases in conjunction with Mass Bank of North America (http://massbank.us) and NIST 20 mass spectral libraries (80 (link)).