Pegasus 4d time of flight mass spectrometer

The replicates of metabolome are n = 10 for all groups. The cecal chyme metabolomic content was determined by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China). The internal standard L-2-chlorophenylalanine (CAS#: 103616-89-3, ≥ 98%) was bought from Hengbai Biotechnology Co., Ltd. (Shanghai, China) and derivatization reagent BSTFA (including 1% TMCS, v/v) was purchased from REGIS Technologies Inc. (Morton Grove, IL, United States). An Agilent 7890 gas chromatograph system combined with a Pegasus 4D time-of-flight mass spectrometer (LECO Corp, St. Joseph, MI, United States) was used for GC/TOFMS analysis. The DB-5MS capillary column was applied in this system (30 m × 250 μm inner diameter, 0.25 μm film thickness (J&W Scientific, Folsom, CA, United States). Analyte injection volume was one μL with splitless mode. The carrier gas was helium, the purge flow at the front inlet was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The initial temperature was kept at 80°C for 1 min, then raised to 290°C at a rate of 10°C/min, then held at 290°C for 12 min. The temperatures in injection, transfer line, and ion source were 280, 295, and 220°C, respectively. In the electron impact mode, the energy was −70 eV. After a 7 min delay in the solvent, mass spectrometry data were acquired at a rate of 12 spectra per second in full-scan mode with the m/z range of 50–600.

GC-MS analysis was performed using a Pegasus 4D time-of-flight mass spectrometer (LECO) equipped with a Gerstel MPS2 autosampler and an Agilent 7890A oven. In the GC step, derivatization products were separated on an Rxi-5Sil^® column (30m, 0.25mm i.d., 0.25µm df) with an IntegraGuard^® pre-column using ultrapure helium at a constant flow of 0.9mL/minute as carrier gas. A split injection and injector port temperature of 240°C were employed, while the transfer line was set at 280°C. A 1µL sample volume was injected at an appropriate split ratio. The linear temperature gradient for GC started with a one-minute hold of the GC column at oven temperature of 70°C, which was then ramped at 10°C/minute to a final temperature of 300°C. Isothermal heating at 300°C was maintained for 5 minutes, followed by a final ramp to initial conditions. In the MS step, mass spectra were obtained using electron impact ionization at 70eV and a scan rate of 17 spectra/sec. Peak alignment and spectrum comparisons were carried out using the Statistical Compare feature of the ChromaTOF^® software (LECO).

The biofluid metabolomics was examined using an Agilent 7890 gas chromatography system equipped with a Pegasus 4D time of flight mass spectrometer (LECO, St. Joseph, MI, USA) [6 (link)]. The tissue metabolomics procedures were performed as follows. First, 100 mg of MG tissue from each sample was added to a 2-mL Eppendorf tube with 0.4 mL of methanol-chloroform (V_methanol: V_chloroform = 3:1) and 30 μL of L-2-chlorophenylalanine (1 mg/mL, stored in dH₂O) and was mixed by vortexing for 10 s. Second, steel balls were placed in the tube and milled for 5 min at 55 Hz. The sample was then centrifuged at 4 °C at 12,000 rpm for 15 min. Third, approximately 0.4 mL of supernatant was transferred into a 2 mL silylated vial. An equal volume (10 μL) of each sample was placed in a new 2 mL silylated vial as a mixed sample for the quality control of the stability of the equipment system, the standard deviation of the beginning, middle and ending retention time of the mixed samples was less than 0.2, which indicates good stability.